JP2013511571A - Imidazo-pyrazoles as GPR119 inhibitors - Google Patents

Abstract

A compound of formula (I) that modulates the activity of G protein-coupled receptor GPR119, wherein X is (A) or (B), Y is O or a bond, and R ¹ is —C ( O) —O—R ³ or R ² is hydrogen, cyano, C ₁ -C ₆ alkyl, or C ₃ -C ₆ cycloalkyl, and R ⁵ is hydrogen, cyano, nitro, C _1- C ₆ fluoroalkyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ fluoroalkoxy, or C ₃ -C ₆ cycloalkyl, R ⁶ is hydrogen, C ₁ -C ₆ alkyl a _C 3 -C ₆ cycloalkyl, -C (O) -NH ₂ or _C 1 -C ₆ alkyl substituted with hydroxy or _C 1 -C ₆ alkoxy,, m is 1 or 2, when m is 1, ^{R 8} is hydrogen C ₁ -C _{6 alkyl, -CH 2 - (C 1 ~C} 5) _{haloalkyl, C} 1 -C ₆ alkyl substituted with C 3 -C ₆ cycloalkyl, or hydroxy, when m is 2 Each R ⁸ is independently C ₁ -C ₃ alkyl or —CH ₂ — (C ₁ -C ₂ ) haloalkyl] and in the treatment of diseases associated with modulation of the G protein-coupled receptor GPR119 in animals. Its use is described herein.
[Chemical 1]

Description

  The present invention relates to a novel class of imidazo-pyrazoles, pharmaceutical compositions containing these compounds, and their use to modulate the activity of the G protein coupled receptor GPR119.

  Diabetes is a disorder where there is a high concentration of blood glucose as a result of abnormal glucose homeostasis. The most common forms of diabetes are type I diabetes (also called insulin dependent diabetes) and type II diabetes (also called non-insulin dependent diabetes). Type II diabetes, which accounts for approximately 90% of all diabetic cases, is associated with microvascular complications (eg, including retinopathy, neuropathy, and kidney damage), and macrovascular complications (eg, accelerated atherosclerosis). Serious progressive disease, including symptom, coronary heart disease, and stroke).

  There is currently no cure for diabetes. Standard treatments for this disease are limited and focus on controlling blood glucose levels to minimize or delay complications. Current therapies target either insulin resistance (metformin or thiazolidinedione) or insulin release from beta cells (sulfonylurea, exanatide). Sulfonylureas and other compounds that work through β-cell depolarization stimulate hypoglycemia because they stimulate insulin secretion independent of circulating glucose concentration. One of the approved drugs, exanatide, stimulates insulin secretion in the presence of high glucose, but lacks oral bioavailability and must be injected. Sitagliptin, a dipeptidyl peptidase IV inhibitor, is a drug that increases the blood concentration of incretin hormone, which can increase insulin secretion and decrease glucagon secretion, and is less well characterized It may have other effects that are not. However, sitagliptin and other dipeptidyl peptidase IV inhibitors can also affect the tissue concentrations of other hormones and peptides, and the long-term consequences of this broader effect have been well investigated. Absent.

  In type II diabetes, muscle cells, adipocytes, and hepatocytes do not respond normally to insulin. This condition (insulin resistance) may be due to a decrease in the number of cellular insulin receptors, disruption of cellular signaling pathways, or both. Initially, beta cells make up for insulin resistance by increasing insulin production. Eventually, however, β cells fail to produce enough insulin to maintain a normal glucose concentration (normoglycemia), indicating progression to type II diabetes.

  In type II diabetes, fasting hyperglycemia occurs due to insulin resistance combined with β-cell dysfunction. There are two modes of abnormal β-cell dysfunction. Namely, 1) increased basal insulin release (which occurs at non-irritating low glucose concentrations) (which is observed not only in type II diabetes but also in the insulin-resistant prediabetic stage of obesity), and 2) hyperglycemic load In response, the insulin release does not increase above the already elevated basal level (this phenomenon does not occur in the pre-diabetic stage and is a precursor to the transition from the insulin-resistant stage of normoglycemia to type II diabetes In some cases). Current therapies treating the latter embodiment include administration of an inhibitor of ATP-sensitive β-cell potassium channel to induce the release of endogenous stored insulin and exogenous insulin. Neither of them achieves accurate normalization of blood glucose concentration, and involves the risk of causing hypoglycemia.

  Therefore, there is great interest in discovering drugs that function in a glucose-dependent manner. Physiological signaling pathways that function in this way are well known, including the intestinal peptides GLP-1 and GIP. These hormones signal through the homologous G protein coupled receptor to stimulate the production of cAMP in pancreatic β cells. Increasing cAMP does not appear to stimulate insulin release during fasting or pre-meal conditions. However, several biochemical targets of cAMP, including ATP-sensitive potassium channels, voltage-sensitive potassium channels, and the exocytosis mechanism, are regulated so that postprandial glucose-stimulated insulin secretion is significantly enhanced. Thus, novel β-cell GPCR agonist modulators, including GPR119, that function similarly also stimulate endogenous insulin release and promote normalization of glucose concentrations in type II diabetic patients. For example, it has also been found that increasing cAMP as a result of stimulation with GLP-1 promotes β-cell proliferation, suppresses β-cell death, and thus improves islet mass. This positive effect on beta cell mass should be beneficial in type II diabetes, where insulin is not fully produced.

  It is well known that metabolic diseases adversely affect other physiological systems, such as complex disease states (eg, “Syndrome X” type I diabetes, type II diabetes, insufficient glucose tolerance, insulin resistance Secondary disease that occurs following diabetes, such as hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, obesity, or cardiovascular disease), or kidney disease or peripheral neuropathy Often accompanied. Thus, there is a need for new treatments for diabetic conditions.

  According to the present invention, a new class of GPR regulators has been discovered. These compounds have the formula I shown below

［式中、
Ｘは、ＡまたはＢ

[Where:
X is A or B

であり、
Ｙは、Ｏまたは結合であり、
Ｒ^１は、−Ｃ（Ｏ）−Ｏ−Ｒ^３または

And
Y is O or a bond;
R ¹ is —C (O) —O—R ³ or

であり、
Ｒ^２は、水素、シアノ、Ｃ_１〜Ｃ_６アルキル、またはＣ_３〜Ｃ_６シクロアルキルであり、
Ｒ^３は、Ｃ_１〜Ｃ_６アルキル、Ｃ_３〜Ｃ_６シクロアルキル、またはＣ_１〜Ｃ_６アルキル、Ｃ_１〜Ｃ_６アルコキシ、Ｃ_１〜Ｃ_６フルオロアルキル、ハロ、もしくはヒドロキシで置換されているＣ_３〜Ｃ_６シクロアルキルであり、但し、ハロ、Ｃ_１〜Ｃ_６アルコキシ、またはヒドロキシ基は、Ｒ^１中のＯに連結している炭素原子では結合しておらず、
Ｒ^４は、Ｃ_１〜Ｃ_６ハロアルキル、Ｃ_１〜Ｃ_６アルキル、ハロ、シアノ、またはＣ_３〜Ｃ_６シクロアルキルであり、
Ｒ^５は、水素、シアノ、ニトロ、Ｃ_１〜Ｃ_６フルオロアルキル、Ｃ_１〜Ｃ_６アルキル、Ｃ_１〜Ｃ_６アルコキシ、Ｃ_１〜Ｃ_６フルオロアルコキシ、またはＣ_３〜Ｃ_６シクロアルキルであり、
Ｒ^６は、水素、Ｃ_１〜Ｃ_６アルキル、Ｃ_３〜Ｃ_６シクロアルキル、−Ｃ（Ｏ）−ＮＨ_２、またはヒドロキシもしくはＣ_１〜Ｃ_６アルコキシで置換されているＣ_１〜Ｃ_６アルキルであり、
Ｒ^７ａおよびＲ^７ｂは、それぞれ独立に、水素、フルオロ、またはＣ_１〜Ｃ_６アルキルであり、
ｍは、１または２であり、ｍが１であるとき、Ｒ^８は、水素、Ｃ_１〜Ｃ_６アルキル、−ＣＨ_２−（Ｃ_１〜Ｃ_５）ハロアルキル、Ｃ_３〜Ｃ_６シクロアルキル、またはヒドロキシで置換されているＣ_１〜Ｃ_６アルキルであり、ｍが２であるとき、各Ｒ^８は、それぞれ独立に、Ｃ_１〜Ｃ_３アルキルまたは−ＣＨ_２−（Ｃ_１〜Ｃ_２）ハロアルキルである］
または薬学的に許容できるその塩によって表すことができる。

And
R ² is hydrogen, cyano, C ₁ -C ₆ alkyl, or C ₃ -C ₆ cycloalkyl,
R ³ is substituted with C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, or C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ fluoroalkyl, halo, or hydroxy C ₃ -C ₆ cycloalkyl, provided that the halo, C ₁ -C ₆ alkoxy, or hydroxy group is not bonded at the carbon atom connected to O in R ¹ ;
R ⁴ is C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkyl, halo, cyano, or C ₃ -C ₆ cycloalkyl,
R ⁵ is hydrogen, cyano, nitro, C ₁ -C ₆ fluoroalkyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ fluoroalkoxy, or C ₃ -C ₆ cycloalkyl. ,
R ⁶ is _hydrogen, C 1 -C _{6 alkyl,} C 3 -C ₆ cycloalkyl, -C (O) -NH ₂ or _C 1 -C ₆ alkyl substituted with hydroxy or _C 1 -C ₆ alkoxy, And
R ^7a and R ^7b are each independently hydrogen, fluoro, or C ₁ -C ₆ alkyl;
m is 1 or 2, and when m is 1, R ⁸ is hydrogen, C ₁ -C ₆ alkyl, —CH ₂ — (C ₁ -C ₅ ) haloalkyl, C ₃ -C ₆ cycloalkyl, or hydroxy a _C 1 -C ₆ alkyl which is substituted, when m is 2, each ^{R 8} is _{independently,} C 1 -C ₃ alkyl or _{-CH 2 - (C 1 ~C 2} ) Is haloalkyl]
Alternatively, it can be represented by a pharmaceutically acceptable salt thereof.

  The compounds of formula I modulate the activity of G protein coupled receptors. More particularly, compounds of formula I modulate GPR119. Therefore, the compounds are useful for the treatment of diseases such as diabetes where GPR119 activity contributes to the pathology or symptoms of the disease. Examples of such conditions include hyperlipidemia, type I diabetes, type II diabetes, idiopathic type I diabetes (type Ib), adult latent autoimmune diabetes (LADA), early-onset type 2 diabetes (EOD) ), Juvenile atypical diabetes (YOAD), juvenile-onset adult diabetes (MODY), malnutrition-related diabetes, gestational diabetes, coronary heart disease, ischemic stroke, restenosis after angioplasty, peripheral vascular disease, intermittent Lameness, myocardial infarction (eg, necrosis and apoptosis), dyslipidemia, postprandial lipemia, impaired glucose tolerance (IGT) condition, fasted plasma glucose glucose condition, metabolic acidosis, ketosis, arthritis, Obesity, osteoporosis, hypertension, congestive heart failure, left ventricular hypertrophy, peripheral arterial disease, diabetic retinopathy, macular degeneration, cataract, diabetic kidney , Glomerulosclerosis, chronic renal failure, diabetic neuropathy, metabolic syndrome, syndrome X, premenstrual syndrome, coronary heart disease, angina, thrombosis, atherosclerosis, transient ischemic attack, stroke Vascular restenosis, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertriglyceridemia, insulin resistance, impaired glucose metabolism, impaired glucose tolerance, abnormal fasting plasma glucose, obesity, erectile dysfunction, Examples include skin and connective tissue disorders, foot ulceration and ulcerative colitis, endothelial disorders, and vasodilatory disorders. The compounds of formula I can also be used for the treatment of neurological disorders such as Alzheimer's disease, schizophrenia, cognitive impairment. The compounds of formula I are also beneficial in gastrointestinal diseases such as inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome. As mentioned above, the compounds of formula I can also be used to stimulate weight loss in obese patients, in particular obese patients suffering from diabetes.

  Another embodiment of this invention is directed to a pharmaceutical composition containing a compound of formula I. Such formulations usually contain a compound of formula I admixed with at least one pharmaceutically acceptable excipient. Such formulations may also contain at least one additional agent (described herein). Examples of such agents include antiobesity agents and / or antidiabetic agents (discussed below). An additional aspect of the invention relates to the use of a compound of formula I in the preparation of a medicament for treating diabetes and related conditions described herein.

FIG. 1 shows the DNA sequence of wild type hGPR119. FIG. 2 is a diagram showing the pFB-VSVS-CMV-polyvector.

  The present invention can be understood more readily by reference to the following detailed description of illustrative embodiments of the invention and the examples contained therein.

  It should be understood that the present invention is not limited to a particular synthetic manufacturing method, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Plural and singular terms should be treated as interchangeable except indicating numbers.

The headings in this document are only used to help readers read the document quickly. These headings should not be construed as limiting the invention or the claims in any way.
a. “Halo” or “halogen” refers to a chlorine, fluorine, iodine, or bromine atom.
b. “Alkyl” refers to a branched or straight chain alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, and the like.
c. “Alkoxy” refers to a straight or branched alkoxy group, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, pentoxy, and the like.
d. “Cycloalkyl” refers to a non-aromatic ring that is fully hydrogenated and exists as a single ring. Examples of such carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
e. “Haloalkyl” refers to a straight or branched alkyl group substituted with one or more halo groups, for example, chloromethane, fluoromethane, dichloromethane, difluoromethane, dibromomethane, trichloromethane, trifluoromethane, chloro Fluoromethane, 1,1,1,2-tetrafluoroethane and the like are indicated.
f. “Fluoroalkyl” refers to a straight or branched alkyl group substituted with one or more fluoro groups, such as fluoromethane, difluoromethane, trifluoromethane, and the like.
g. “Haloalkoxy” means a straight or branched alkoxy group substituted with one or more halo groups, for example, chloromethoxy, fluoromethoxy, dichloromethoxy, difluoromethoxy, dibromomethoxy, trichloromethoxy, trifluoro It refers to methoxy, chlorofluoromethoxy, 1,1,1,2-tetrafluoroethoxy and the like.
h. “Therapeutically effective amount” means (i) treating or preventing a particular disease, condition or disorder, (ii) alleviating and ameliorating one or more symptoms of a particular disease, condition or disorder, Or (iii) means the amount of a compound of the invention that prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein.
i. “Patient” refers to warm-blooded animals such as guinea pigs, mice, rats, gerbils, cats, rabbits, dogs, monkeys, chimpanzees, and humans.
j. “Treat” refers to the ability of a compound to reduce, eliminate or slow the progression of a patient's disease (or condition) or any tissue damage associated with the disease.
k. The terms “modulated”, “modulating”, or “modulate”, as used herein, unless otherwise indicated, refer to activation of the G protein-coupled receptor GPR119 using a compound of the present invention. .
l. “Pharmaceutically acceptable” means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or the mammal to be treated therewith. Indicates.
m. “Salt” shall refer to pharmaceutically acceptable salts and salts suitable for use in industrial processes such as the preparation of compounds.
n. "Pharmaceutically acceptable salt" shall refer to either a pharmaceutically acceptable acid addition salt or a "pharmaceutically acceptable base addition salt" depending on the actual structure of the compound.
o. "Pharmaceutically acceptable acid addition salt" shall correspond to any non-toxic organic or inorganic acid addition salt of the compound represented by formula I or any of its intermediates. Illustrative inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. . Illustrative organic acids that form suitable salts include mono, di, and tricarboxylic acids. Examples of such acids are, for example, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, Benzoic acid, hydroxy-benzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, 2-phenoxybenzoic acid, p-toluenesulfonic acid, and sulfonic acids such as methanesulfonic acid and 2-hydroxyethanesulfonic acid. Such salts may exist in either a hydrated or substantially anhydrous form. In general, acid addition salts of such compounds are soluble in water and various hydrophilic organic solvents.
p. "Pharmaceutically acceptable base addition salt" shall correspond to any non-toxic organic or inorganic base addition salt of the compound represented by formula I or any of its intermediates. Examples of bases that form suitable salts include alkali metal or alkaline earth metal hydroxides such as sodium hydroxide, potassium, calcium, magnesium, barium; ammonia; and methylamine, dimethylamine, trimethylamine, picoline, and the like , Aliphatic, cycloaliphatic, or aromatic organic amines.
q. “Compounds of formula I”, “compounds of the invention”, and “compounds” are used interchangeably throughout the application and should be treated as synonyms.
“Isomers” means “stereoisomers” and “geometric isomers” as defined below.
r. “Stereoisomer” means a compound having one or more chiral centers, and each center may exist in the R or S configuration. Stereoisomers include all diastereoisomers, enantiomers, and epimeric forms, and racemates and mixtures thereof.
s. “Geometric isomer” means compounds that may exist in the form of cis, trans, anti, syn, entgegen (E), and tsuzanmen (Z), and mixtures thereof.

  The compounds of the present invention contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. Unless otherwise specified, all stereoisomeric forms of the compounds of the invention, as well as mixtures thereof, including racemic mixtures, are intended to form part of the invention. In addition, the present invention encompasses all geometric and positional isomers. For example, if a double bond or fused ring is incorporated into a compound of the invention, both cis and trans forms and mixtures are encompassed within the scope of the invention.

  Diastereoisomer mixtures are separated into their individual diastereoisomers based on their physicochemical differences by methods well known in the art such as chromatography and / or fractional crystallization, distillation, sublimation, etc. be able to. Enantiomers convert an enantiomeric mixture into a diastereoisomeric mixture by reaction with a suitable optically active compound (eg, a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride). Isomers can be separated and separated by converting (eg, hydrolyzing) the individual diastereoisomers to the corresponding pure enantiomers. Also, some of the compounds of the present invention may be atropisomers (eg, substituted biaryls) and are considered as part of this invention. Enantiomers can also be separated using chiral HPLC (high pressure liquid chromatography) columns.

  It is contemplated that intermediates and compounds of the present invention may exist in different tautomeric forms, and all such forms are embraced within the scope of the invention. The term “tautomer” or “tautomeric form” refers to structural isomers of different energies that are interconvertible with a low energy barrier. For example, proton tautomers (also known as prototrophic tautomers) involve interconversion by proton transfer, such as keto-enol isomers and imine-enamine isomers. A specific example of a proton tautomer is an imidazole moiety that allows protons to move between two ring nitrogens. Valence tautomers involve interconversion by reorganization of some of the bonding electrons. The equilibrium between the closed and open forms of some intermediates (and / or mixtures of intermediates) is reminiscent of the rotatory process involving aldoses known to those skilled in the art.

  In addition, the compounds of the present invention may exist in unsolvated forms or in solvated forms with pharmaceutically acceptable solvents such as water and ethanol. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention. A compound may exist in one or more crystalline states, ie, polymorphs, or may exist as an amorphous solid. All such forms are encompassed by the claims.

The present invention is the same as listed herein except that one or more atoms are replaced with atoms whose atomic mass or mass number is different from the atomic mass or mass number normally found in nature. Also included are isotopically-labeled compounds of the present invention. Examples of isotopes that can be incorporated into the compounds of the present invention include ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P. , ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I, ³⁶ Cl and the like, respectively, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine.

Certain isotopically-labelled compounds of the present invention (eg, those labeled with ³ H and ¹⁴ C) are useful in compound and / or substrate tissue distribution assays. Tritiated isotopes (ie, ³ H) and carbon-14 isotopes (ie, ¹⁴ C) are particularly preferred because they are readily prepared and detectable. Furthermore, certain therapeutic benefits that result from greater metabolic stability, such as replacement with heavier isotopes such as deuterium (ie ² H) (eg, increased in vivo half-life or reduced dosage requirements) May be provided and therefore may be preferred in certain circumstances. Positron emitting isotopes such as ¹⁵ O, ¹³ N, ¹¹ C, ¹⁸ F are useful in Positron Emission Topography (PET) studies to examine substrate occupancy. Isotopically-labeled compounds of the invention are generally disclosed herein below in the schemes and / or examples by using isotope-labeled reagents instead of non-isotopically-labeled reagents. Can be prepared according to procedures similar to those described above.

  Some of the compounds of Formula I contain a 3-oxa-7-azabicyclo [3.3.1] nonane ring attached to the pyrimidine ring via an ether linkage, as illustrated below. The azabicyclo-nonane exists as a geometric isomer and can exist as either the syn or anti isomer illustrated below.

In one embodiment of compounds having Formula I, X is A and R ¹ is —C (O) —O—R ³ .

In another embodiment of the compounds having formula I, R ⁶ and R ⁸ are each hydrogen.

In another embodiment of the compounds having formula I, R ³ is C ₃ -C ₆ cycloalkyl substituted with C ₁ -C ₃ alkyl.

In another embodiment of the compounds having Formula I, R ^7a and R ^7b are each independently hydrogen, fluoro, or C ₁ -C ₃ alkyl.

In another embodiment of the compounds having formula I, R ² is hydrogen and R ⁵ is C ₁ -C ₆ alkyl.

Synthesis The compounds of the present invention can be synthesized by synthetic routes that include methods similar to those well known in the chemical art, particularly in light of the description contained herein. Starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wis.), Or are readily prepared using methods known to those skilled in the art (eg, Louis F. Fieser and From Mary Fieser, Reagents for Organic Synthesis, Volumes 1-19, Wiley, New York (1967-1999), or Beilsteins Handbuch der organischen Chemie, 4th edition, Berlin, published by Spring-Verlag. Prepared by the methods generally described in the

  For illustrative purposes, the reaction scheme illustrated below illustrates potential routes to synthesize the compounds of the invention as well as key intermediates. See the Examples section below for a more detailed description of the individual reaction steps. One skilled in the art will appreciate that other synthetic routes may be used to synthesize the compounds of the invention. Detailed starting materials and reagents are shown in the scheme and discussed below, but can be readily replaced with other starting materials and reagents to provide various derivatives and / or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

  Compounds of formula I can be prepared using methods of ether formation that are also known in the art. 1) Hughes, D .; L. Organic Reactions 1992, 42 335-656, Hoboken, NJ, USA 2) Tikad, A .; Routier, S .; Aksira, M .; Leger, J .; -M. l, Jarry, C .; Guillaumet, G .; Synlett 2006, 12, 1938-42, and 3) Loksha, Y .; M.M. Globisch, D .; Pedersen, E .; B. La Colla, P .; Collu, G .; Roddo, R .; J. et al. Het. Chem. Look to textbooks that describe these reactions in more detail, such as 2008, 45, 1161-6.

Scheme I immediately below illustrates an alternative method for assembling compounds of formula I. The central part of the molecule is an optionally substituted pyrimidine ring. Compounds of formula I are produced by forming both ether and amino bonds with pyrimidines as shown below. The order in which this reaction sequence is carried out is not important, except when R ⁵ is cyano or nitro. In such cases, Steps IB and IC are used to assemble the compound of Formula I.

The starting material of Reaction Scheme I is a dihydroxy-pyrimidine of Structural Compound I-1 in which R ² and R ⁵ are typically represented by the same substituents as desired in the final product as described herein. It is. Such methods for producing pyrimidines are known in the art.

The chlorination reaction of Step IA is carried out as known in the art. The compound of structure I-1 is used in excess, with or without additives such as triethylamine, N, N-dimethylaniline, N, N-diisopropylethylamine, or solvents such as toluene, benzene, xylene React with a chlorinating reagent such as POCl ₃ (phosphorus oxychloride) used in (Matulenko, MA, et al., Bioorg. Med. Chem. 2007, 15, 1586-1605). This reaction can be carried out at a temperature ranging from room temperature (about 23 ° C.) to about 140 ° C., depending on the choice of conditions. Alternative chlorinating reagents can consist of PCl ₃ (phosphorus trichloride), POCl ₃ / PCl ₅ (phosphorus pentachloride), thionyl chloride, oxalyl chloride, or phosgene to give the dichloropyrimidine of structure I-2. In some cases, the dichloropyrimidine of structure I-2 can also be obtained from commercial sources. Optionally, the dichloropyrimidine of structure I-2 may be isolated and recovered from the reaction and further purified as is known in the art. Alternatively, unpurified material may be used in Step IB described below.

In Step I-B of Scheme I, an amino bond is formed between the imidazo-pyrazole of structure I-3 and the dichloropyrimidine of structure I-2. In the imidazo-pyrazole structure I-3, R ⁶ and R ⁸ are typically represented by the same substituents as desired in the final product, as described herein. Such imidazo-pyrazole derivatives are known in the literature or can be conveniently prepared by various methods well known to those skilled in the art (US2989537; Anti-Cancer Drug Des. 1987, 2,235). ). The amino bond to produce I-5 is formed by reacting equal amounts of compounds of structures I-2 and I-3 in a solvent in the presence of a base. A set of conditions for this transformation is about 0-120 depending on which solvent is used in structures I-2 and I-3 in polar protic solvents such as ethanol, propanol, isopropanol, butanol and the like. The reaction is carried out at a temperature in the range of 0C for 0.5 to 24 hours. A typical condition utilized for this reaction is the use of isopropanol as a solvent heated at 108 ° C. for 1 hour. Alternatively, an amine base such as triethylamine or diethylisopropylamine, or an inorganic base such as sodium bicarbonate, potassium carbonate, sodium carbonate may be added to the reaction. When one of the above amines or inorganic bases is used, the solvent is a polar aprotic solvent such as acetonitrile, N, N-dimethylformamide (“DMF”), tetrahydrofuran (“THF”), 1,4-dioxane ( About 0 to 100 ° C. and 0.5 to 24 hours). Typical conditions utilized for this reaction include the use of diethyl isopropylamine in acetonitrile for 3 hours at room temperature. The use of hydrochloric acid in polar protic solvents alone or in combination such as water, methanol, ethanol, propanol, etc. can also be used for this conversion at temperatures of about 0-110 ° C. Typical conditions are those using water at 78 ° C. in ethanol. A preferred method of producing I-5 is by reacting structures I-2 and I-3 with sodium bis (trimethylsilyl) amide in tetrahydrofuran. The intermediate of structure I-5 can be isolated and recovered from the reaction and further purified as is known in the art. Alternatively, unpurified material may be used in Steps IC described below.

In Step I-C of Scheme I, an ether bond is formed between the intermediate of structure I-5 and the alcohol of structure I-4 to produce a compound of formula I. In the alcohol of structure I-4, X is A, B or C, and R ^7a and R ^7b are represented by the same substituents as found in the desired end product. The substituent represented by R ¹ can be manipulated after generating the nucleus of formula I. Such variations are well known to those skilled in the art and should be considered part of this invention. In Step IC, sodium hydride; sodium and sodium in a solvent such as DMF, THF, 1,2-dimethoxyethane, 1,4-dioxane, N, N-dimethylacetamide, dimethyl sulfoxide (“DMSO”) Equivalent reactants are reacted in the presence of a base such as potassium tert-butoxide; sodium, potassium, and lithium bis (trimethylsilyl) amide; sodium, potassium, and lithium tert-amyl oxide. Typical conditions for this transformation include the use of sodium bis (trimethylsilyl) amide in 1,4-dioxane at about 105 ° C. for 1 hour.

  After the reaction is complete, the desired compound of formula I can be recovered and isolated as is known in the art. The compound can be recovered by evaporation, extraction, etc., as is known in the art. The compound may optionally be purified by chromatography, recrystallization, distillation, or other techniques known in the art.

In an alternative synthesis shown above in Reaction Scheme I, a dichloro-pyrimidine of structure I-2 is first reacted with an alcohol of structure I-4 to produce the intermediate shown by structure I-6. As in step IC, structure I-4 is an alcohol where X is A, B or C, depending on the desired end product. In these heterocycles, R ¹ and R ⁴ will usually be represented by the same substituents as desired in the final product, or R ¹ may be manipulated after generating the nucleus of formula I. it can.

  Equivalent compounds of structure I-2 and structure I-4 are reacted in the presence of a polar aprotic solvent and a base to produce the intermediate of structure I-6 shown in Step ID. As a suitable system, sodium hydride, sodium and sodium hydride in a solvent such as DMF, THF, 1,2-dimethoxyethane, 1,4-dioxane, N, N-dimethylacetamide, DMSO at a temperature of 0-140 ° C. Examples include potassium tert-butoxide, sodium, potassium, and lithium bis (trimethylsilyl) amide; bases such as sodium, potassium, and lithium tert-amyl oxide. Typical conditions for this conversion include the use of potassium tert-butoxide in THF at about 0 ° C. to room temperature for 14 hours. The intermediate of structure I-6 can be isolated and recovered from the reaction and further purified as is known in the art. Alternatively, unpurified material may be used in Step IE described below.

  The intermediate of structure I-6 can then be reacted with the imidazo-pyrazole derivative I-3 previously described above to produce a compound of formula I. Usually, an equivalent amount of an imidazo-pyrazole of structure I-3 is reacted with a chloro intermediate of formula I-6 in the presence of a base. Suitable bases are sodium hydride, sodium or potassium tert in a solvent such as DMF, THF, 1,2-dimethoxyethane, 1,4-dioxane, N, N-dimethylacetamide, or DMSO, or mixtures thereof. -Butoxide, sodium, potassium, or lithium bis (trimethylsilyl) amide; and sodium, potassium, or lithium tert-amyl oxide. These reactions can be carried out at temperatures in the range of about −10 to 150 ° C., depending on the solvent used. The reaction is usually allowed to proceed for a time in the range of about 15 minutes to 24 hours in an inert atmosphere. Suitable conditions include sodium bis (dimethylsilyl) amide in 1,4-dioxane at 105 ° C. for 1 hour.

  Alternatively, this reaction may involve the intermediate of structure I-6 and the imidazo-pyrazole derivative of structure I-3 in a polar protic solvent such as methanol, ethanol, propanol, isopropanol, butanol for 0.5-24 hours. You may implement by heating. Typical conditions for this conversion are heating in isopropanol at 108 ° C. for 2 hours.

This reaction can also be carried out using a transition metal catalyst for the formation of important substituted amine bonds found in compounds of formula I. Transition metal catalysts include, but are not limited to, triphenylphosphine) palladium (Pd (PPh ₃ ) ₄ ), palladium chloride (II) (PdCl ₂ ), palladium acetate (II) (Pd (OAc) ₂ ), (Tris ( Dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃ ), copper (I) iodide (CuI), copper (II) acetate (Cu (OAc) ₂ ), and copper (II) trifluoromethane (Cu) (OTf) ₂ ) Usually, these reactions utilize a base, and suitable bases for use with a palladium catalyst are 1,4-dioxane, THF, 1,2-dimethoxyethane. Sodium tert-butoxide, potassium tert-butoxide, potassium tert-amyl oxide in a suitable solvent such as toluene Or can be K ₃ PO _4. For the use of a copper catalyst, a suitable base consists of an alkaline base such as sodium carbonate, potassium carbonate, cesium carbonate in a suitable solvent such as DMF, DMSO, dimethylacetamide. Can be.

Usually, when a ligand is added, an amine formation reaction can be promoted. The ligand for the reaction catalyzed by palladium is not limited, but 9,9-dimethyl-4,5-bis (diphenylphosphino) xanthene (xanthophos), 2,2′-bis (diphenylphosphino) ) -1,1′-binaphthyl (BINAP), 1,1′-bis (diphenylphosphino) ferrocene (DPPF), 2,8,9-triisobutyl-2,5,8,9-tetraaza-1-phos Fabicyclo [3.3.3] undecane (P [N (i-Bu) CH ₂ CH ₃ ] ₃ N), tri-tert-butylphosphine (tert-Bu ₃ P), (biphenyl-2-yl) bis (Tert-Butyl) phosphine (JohnPhos), Pd-PEPPSI ™ -SIPr: (1,3-bis (2,6-diisopropylphenyl) -4,5-dihydride Imidazol-2-ylidene) (3-chloropyridyl) can be mentioned palladium (II) dichloride. Examples of the ligand suitable for the reaction using copper as a catalyst include, but are not limited to, L-proline, N-methylglycine, and diethylsalicylamide. A suitable condition for the production of the compound of formula I is the use of Pd ₂ (dba) ₃ in toluene with sodium tert-butoxide at 120 ° C. for 12 hours.

  After completing the reaction, the desired compound of formula I can be recovered and isolated as is known in the art. The compound can be recovered by evaporation, extraction, etc. as is known in the art. The compound may optionally be purified by chromatography, recrystallization, distillation, or other techniques known in the prior art.

Also, as will be readily appreciated by those skilled in the art, many of the substituents represented by R ¹ can be manipulated after generating the nucleus of formula I. Such variations are well known to those skilled in the art and should be considered part of this invention.

Scheme II describes a method for producing alcohols of structures II-14 and II-15 corresponding to X = B in formula I of the present invention. R ³ , R ⁶ , R ^7a and R ^7b are typically represented by the same substituents as desired in the final product, as described herein. Synthesis of compounds of structure II-8 from compounds of structure II-7 is known in the art. Their transformation (step II-A) is taught in the literature and is described in J. Org. Org. Chem. 1981, 46, 3196-3204, JP2009090744, WC0535303, J. MoI. Am. Chem. Soc. 2008, 130, 5654-5655, and Org. Lett. 2006, 3, 430-436. In Step II-B of Scheme II, a standard protocol known in the art, such as using sodium borohydride in an alcoholic solvent such as methanol at a temperature in the range of about 0 ° C. to room temperature. To reduce the ketone carbonyl group. Step II-D, ie, removing the benzyl protecting group from structure II-10 to give II-11 can be achieved by hydrogenolysis. Typical conditions for this reaction include the use of hydrogen and palladium catalysts such as 5-20% palladium on carbon and 10-20% palladium hydroxide. Typical solvents for this reaction are ethanol, methanol, tetrahydrofuran, or ethyl acetate.

  If a pyrimidine substituent is desired in the final product, in a protic solvent such as ethanol or methanol, or in a polar aprotic solvent such as 1,4-dioxane, tetrahydrofuran, N, N-dimethylformamide or dimethyl sulfoxide In the presence of a base such as cesium carbonate or N, N-diisopropylethylamine, compound II-11 is added to the appropriately substituted 2-chloropyrimidine shown as structure II-12 to give structure II-14. Can be generated. Such reactions can be carried out at temperatures ranging from about room temperature to about 110 ° C. Alternatively, structures II-11 and II can be used without the use of a solvent, in the presence of a base such as N, N-diisopropylethylamine, or when compound II-11 is used in excess, without the use of a base or solvent. -12 compounds can also be heated together.

If a carbamate substituent is desired in the final product, an equivalent amount of an alkylhaloform of structure II-13 in dichloromethane or chloroform in the presence of a base such as N, N-diisopropylethylamine, triethylamine, pyridine, etc. The mate is reacted with a compound of structure II-11. Alternatively, di-tert-butyl dicarbonate (BOC anhydrous) in the presence of an amine base such as N, N-diisopropylethylamine, pyridine, 2,6-lutidine, or triethylamine in a solvent such as dichloromethane, chloroform, or tetrahydrofuran. Or a dialkyl dicarbonate such as diisopropyl dicarbonate can be used to produce a compound of structure II-15 from a compound of structure II-11. In addition, when R ³ = 1-methyl-cyclopropyl or 1-difluoromethyl-cyclopropyl, in a solvent such as dichloromethane, dichloroethane, dimethoxyethane, tetrahydrofuran, etc., triethylamine, N, N-diisopropylethylamine, etc. Carbamate functional groups can be introduced using carbonate II-13 ′ (see WO09105717 and WO09005677) at temperatures ranging from about 0 ° C. to about ambient temperature in the presence of such bases.

  The final structure II-14 or II-15 can be isolated and purified as is known in the art. If desired, this final structure can be subjected to a separation step to obtain the desired syn or anti isomer.

Alternatively, via a double Mannich reaction between the bis-aminol ether derivative II-9 and the ketone II-7, the ketone carbonyl is then reduced and the functional group is manipulated to obtain the structure of type II-10. Thus, an asymmetric structure of formula II-10 can be reached in which at least one of R ^7a and R ^7b is hydrogen. It will be appreciated that in certain instances, R ^9a is preferably an α-methyl-benzyl group rather than the benzyl group shown in structure II-10. Suitable R ^9b groups include methyl or ethyl. The use of the double Mannich reaction to obtain the structure of formula II-8 has been published in the chemical literature (Tetrahedron 2005 61, 5876-5888; Org. Lett. 2006 8, 3399-3401) and has been realized by those skilled in the art. can do. Similarly, starting from readily available starting materials, using protocols known in the chemical literature for the production of cis- or trans-difluoro-2,6 cyclohexanone (Tetrahedron 1970 26, 2447), R ^7a and It is also possible to arrive at the structure of formula II-10 where R ^7b is both fluoro.

  Scheme III describes the preparation of compounds of formula III-19 corresponding to X = A in formula I.

As shown in Scheme III, compounds of formula III-19 wherein R ³ is as described herein and at least one of R ^7a and R ^7b is hydrogen are commercially available N-tert-butoxycarbonyl It can be prepared starting from -4-piperidone (Aldrich) or from 4-piperidone followed by carbamate preparation. Compounds of formula III-19 are prepared by reduction of compounds of formula III-16 or III-18 by reduction of ketone carbonyl, as shown by step III-A. Suitable conditions for this include the use of sodium borohydride in a mixture of an alcoholic solvent such as ethanol and THF. Compounds of formula III-19 in which at least one of R ^7a and R ^7b is fluoro are described in J. Am. Org. Chem. 2003 68, 3232 and J.M. Org. Chem. 2002 67, 8610 can be prepared by enolizing a ketone and trapping it as a silyl enol ether and reacting with a suitable electrophilic fluoro source. Compounds of formula III-18 in which at least one of R ^7a and R ^7b is an alkyl group can be similarly prepared using a suitable electrophilic alkyl group such as an alkyl halide or alkyl sulfonate. In addition, structures of formula III-19, where R ^7a and R ^7b are both halo such as fluoro, are readily available from N-tert-butoxycarbonyl-4-piperidone, as well known in the chemical literature. (Tetrahedron, 1970, 26, 2447). When X is A in the formula I of the present invention, such piperidine compounds are either commercially available, known in the literature, or commercially available (Aldrich) N-tert-butoxycarbonyl-4-piperidone or suitable It is also recognized that it can be prepared from other piperidones that are N-protected.

The tert-butyloxycarbonyl group (R ³ is tert-butyl) can be removed using acids such as hydrochloric acid and trifluoroacetic acid at many stages of the synthesis, and the resulting free amine is It will be appreciated that the general conditions described in Scheme II Steps II-E ′ and II-E, respectively, can be used to convert to another carbamate or pyrimidine. The preparation of compounds of formula III-19 is also described in WO2009014149.

Scheme IV describes the synthesis of compounds of formula IV-23. Compounds of formula IV-23 can be prepared by route A in Scheme IV, according to examples in the chemical literature, by a chemical engineer. In route A, a hydrazine derivative IV-1 (1-hydrazino-2-propanol (CAS number 18501-20-7), 2-hydroxyethyl hydrazide (CAS number 109-84-) in an alcohol solvent such as ethanol or isopropanol is used. 2) by condensation of a cyclocondensation (step IV-3) of 2-hydrazino-1-propanol (J. Am. Chem. Soc. 1954, 76, 1283, etc.) and the appropriate α-cyanoaldehyde IV-2 The aminopyrazole derivative IV-4 is formed. Compounds of formula IV-2 can be obtained by deprotecting alkyl or aryl nitriles followed by quenching with ethyl formate (see J. Med. Chem. 1982, 25, 235-242; WO2007099323). ). Treatment of the intermediate of formula IV-4 with sulfuric acid (step IV-5) (US 2995937; Anti-Cancer Drug Des. 1987, 2, 235) results in the compound of formula IV-23 by dehydration cyclization. Alternatively, an intermediate of formula IV-23 can be obtained from IV-4 by acylation or sulfonation of the amine, activation of N-1 hydroxylethyl by sulfonyl formation, ring closure, and removal of the amine masking group. (Anti-Cancer Drug Des. 1987, 2, 235; EP332156). The compound of formula IV-23, wherein R ⁶ is hydrogen, is obtained by first converting the hydrazino alcohol of formula IV-1 to commercially available 2-ethoxymethylenemalononitrile (CAS number 123-06-8), ie R ⁹ = CN. By condensation with IV-6, or commercially available (ethoxymethylene) ethyl cyanoacetate (CAS number 94-05-3), ie IV-6 where R ⁹ = CO ₂ Et, to give intermediate IV-8 Can be synthesized. A substrate of formula IV-8 in which R ⁹ = CN is subjected to the same conditions as in step IV-5 to give an imidazo-pyrazole of formula IV-23. For compounds of formula IV-8 where R ⁹ = CO ₂ Et, the substrate can be hydrolyzed under basic conditions to the carboxylic acid and then subjected to step IV-5.

Another approach to the imidazo-pyrazoles of formula IV-23 where R ⁸ is a C ₁ -C ₆ substituted alkyl, aryl, and heteroaryl derivative is shown above in Route C. Treatment of an acetylenic nitrile of the formula IV-9 with hydrazine (step IV-10) gives the enamine nitrile derivative IV-11, which is cyclized to give the aminopyrazole derivative IV-12 (Tet. Lett. 2008, 49, 3104; J. Chem. Soc., Perkin Trans. 1, 1981, 2997). Treatment of aminopyrazole derivative IV-12 with 1,2-dibromoethane, base, and heat in an organic solvent in the same manner as EP332156 results in IV-23.

  As will be readily appreciated by those skilled in the art, it may be necessary to protect the distal functional groups (eg, primary or secondary amines) of the intermediate. The need for such protection will vary depending on the nature of the distal functional group and the conditions of the preparation methods. Suitable amino protecting groups (NH-Pg) include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Similarly, “hydroxy protecting group” refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable hydroxyl protecting groups (O—Pg) include, for example, allyl, acetyl, silyl, benzyl, para-methoxybenzyl, trityl and the like. The need for such protection is readily determined by one skilled in the art. For a review on protecting groups and their uses, see T.W. W. See Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

  As noted above, some of the compounds of the present invention can form salts with pharmaceutically acceptable cations. Some of the compounds of the present invention can form salts with pharmaceutically acceptable anions. All such salts are within the scope of the present invention, and the acidic and basic entities are usually separated by conventional methods, eg, in an aqueous, non-aqueous, or partially aqueous medium, as appropriate. Can be prepared by combining them in a stoichiometric ratio. The salt is recovered by filtration, precipitating with a non-solvent, filtration after evaporation, evaporation of the solvent, or lyophilization in the case of an aqueous solution. The compound is obtained in crystalline form according to procedures known in the art, such as dissolving in a suitable solvent (s) such as ethanol, hexane, water / ethanol mixtures.

The present invention is identical to those listed herein except that one or more atoms are replaced with atoms whose atomic mass or mass number is different from the atomic mass or mass number normally found in nature. Also included are isotopically-labeled compounds of the present invention. Examples of isotopes that can be incorporated into the compounds of the present invention include ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P. , ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I, ³⁶ Cl, and the like, respectively, are the isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine.

Certain isotopically-labeled compounds of the present invention (eg, compounds labeled with ³ H and ¹⁴ C) are useful in compound and / or substrate tissue distribution assays. Certain isotopically-labeled ligands incorporating tritium, ¹⁴ C, ³⁵ S, and ¹²⁵ I could be useful in radioligand binding assays. Tritiated (ie, ³ H) and carbon-14 (ie, ¹⁴ C) isotopes are particularly preferred because they are easy to prepare and have good detectability. In addition, substitution with heavier isotopes such as deuterium (ie ² H) may lead to certain therapeutic advantages that result from higher metabolic stability (eg, increased in vivo half-life or reduced dosage requirements). ) And thus may be preferred in some circumstances. Positron emitting isotopes such as ¹⁵ O, ¹³ N, ¹¹ C, ¹⁸ F are useful in Positron Emission Topography (PET) studies for examining receptor occupancy. The isotopically-labeled compounds of the present invention generally follow a procedure similar to that disclosed in the schemes and / or examples below, using isotope-labeled reagents instead of non-isotopically labeled reagents. Can be prepared.

  Certain compounds of the present invention may exist in more than one crystal form (commonly referred to as “polymorphs”). Polymorphs can be obtained by crystallization under various conditions, for example, during crystallization, different recrystallization solvents or solvent mixtures, crystallization at different temperatures, and / or ultra-fast to ultra-slow cooling. A range of different cooling modes can be used to prepare. Polymorphs can also be obtained by heating or melting a compound of the invention and then slowly or rapidly cooling it. The presence of polymorphs can be determined by solid probe NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, powder X-ray diffraction, or other such techniques.

Medical Use The compounds of the present invention modulate the activity of the G protein coupled receptor GPR119. Therefore, the compound is useful for the prevention and treatment of diseases such as diabetes in which the activity of GPR119 contributes to the pathology or symptoms of the disease. Accordingly, another aspect of the present invention is a method of treating metabolic diseases and / or metabolism related disorders in an individual, wherein an individual in need of such treatment is provided with a therapeutically effective amount of a compound of the present invention, It includes a method comprising administering a salt of a compound, or a pharmaceutical composition containing such a compound. Metabolic diseases and metabolism-related disorders include, but are not limited to, hyperlipidemia, type I diabetes, type II diabetes, idiopathic type I diabetes (type Ib), adult latent autoimmune diabetes (LADA), early onset Type 2 diabetes (EOD), juvenile atypical diabetes (YOAD), juvenile-onset adult diabetes (MODY), malnutrition-related diabetes, gestational diabetes, coronary heart disease, ischemic stroke, restenosis after angioplasty, Peripheral vascular disease, intermittent claudication, myocardial infarction (eg, necrosis and apoptosis), dyslipidemia, postprandial lipemia, impaired glucose tolerance (IGT) state, fasting plasma glucose abnormal state, metabolic acidosis, ketosis, arthritis Obesity, osteoporosis, hypertension, congestive heart failure, left ventricular hypertrophy, peripheral arterial disease, diabetic retinopathy, macular degeneration, cataract, diabetic nephropathy, glomerulosclerosis, chronic renal failure, diabetic Neuropathy, metabolic syndrome, syndrome X, premenstrual syndrome, coronary heart disease, angina, thrombosis, atherosclerosis, myocardial infarction, transient ischemic attack, stroke, vascular restenosis, hyperglycemia, high Insulinemia, hyperlipidemia, hypertriglyceridemia, insulin resistance, impaired glucose metabolism, impaired glucose tolerance, abnormal fasting plasma glucose, obesity, erectile dysfunction, skin and connective tissue disorders, foot injury Selected from ulceration, endothelial damage, hyper apo B lipoproteinemia, and vascular extensibility disorders. Furthermore, the compounds of the present invention can also be used for the treatment of neurological disorders such as Alzheimer's disease, schizophrenia and cognitive impairment. The compounds of the present invention are also beneficial in gastrointestinal diseases such as inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome. As mentioned above, the compounds of the invention can also be used to stimulate weight loss in obese patients, particularly obese patients suffering from diabetes.

  In accordance with the foregoing, the present invention further provides a method of preventing or ameliorating a symptom of any of the aforementioned diseases or disorders in a subject in need thereof, comprising a therapeutically effective amount of a compound of the present invention. A method comprising administering to a subject is provided. Another aspect of the invention involves the preparation of a medicament for treating diabetes and its associated complications.

  In order to exhibit the above therapeutic properties, the compound needs to be administered in an amount sufficient to modulate the activation of the G protein coupled receptor GPR119. This amount can vary depending on the particular disease / condition being treated, the severity of the patient's disease / condition, the patient, the particular compound being administered, the route of administration, and the presence of other underlying conditions within the patient, and the like. When administered systemically, the compound typically exhibits its effect in a dosage range of about 0.1 mg / kg / day to about 100 mg / kg / day for any of the diseases or conditions listed above. Daily continuous administration may be desirable and will depend on the conditions outlined above.

  The compounds of the present invention can be administered by a variety of routes. The compounds of the present invention can be administered orally. The compounds of the present invention can also be administered parenterally (ie, subcutaneously, intravenously, intramuscularly, intraperitoneally, or intrathecally), rectally, or topically.

Co-administration The compounds of the invention can also be used with other agents for treating the diseases, conditions, and / or disorders described herein. Accordingly, there is also provided a method of treatment comprising administering a compound of the present invention in combination with other agents. Suitable drugs that can be used in combination with the compounds of the present invention include anti-obesity drugs (including appetite suppressants), anti-diabetic drugs, anti-hyperglycemic agents, anti-hyperlipidemic drugs, And antihypertensive drugs.

  Suitable antidiabetic agents include acetyl-CoA carboxylase 2 (ACC-2) inhibitors, diacylglycerol O-acyltransferase 1 (DGAT-1) inhibitors, phosphodiesterase (PDE) 10 inhibitors, sulfonylureas (eg, acetohexa Mido, Chlorpropamide, Diabines, Glibenclamide, Glipizide, Glyburide, Glimepiride, Gliclazide, Glipentide, Glyquidone, Glysolamide, Tolazamide, Tolbutamide, Meglitinide, α-Amylase inhibitors (eg, tendamistat) tendamistat), trestatin, and AL-3688), alpha-glucoside hydrolase inhibitors (eg acarbose), alpha-glucosidase Pesticides (eg, adiposine, camiglybose, emiglitate, miglitol, voglibose, prazimicin Q, and salvostatin), PPARγ agonists (eg, valaglitazone, ciglitazone, darglitazone, isglitazone, isaglitazozone, isaglitazozone, Rosiglitazone, and troglitazone), PPARα / γ agonists (eg, CLX-0940, GW-1536, GW-1929, GW-2433, KRP-297, L-79449, LR-90, MK-0767, and SB- 219994), biguanides (eg, metformin), glucagon-like peptide 1 (GLP-1) agonists (eg, exendin 3 and exendin 4) Protein tyrosine phosphatase 1B (PTP-1B) inhibitors (eg, Troduschemine, hirtiosal extract, and Zhang, S. et al., Drug Discovery Today, 12 (9/10), 373-381) ), SIRT-1 inhibitors (eg, reservatrol), dipeptidyl peptidase IV (DPP-IV) inhibitors (eg, sitagliptin, vildagliptin, alogliptin, and saxagliptin), insulin secretion Stimulant, fatty acid oxidation inhibitor, A2 antagonist, c-jun amino terminal kinase (JNK) inhibitor, insulin, insulin mimetic, glycoge Phosphorylase inhibitors, VPAC2 receptor agonists, and SGLT2 inhibitors (sodium-dependent glucose transporter inhibitors such as dapagliflozin). Preferred antidiabetic agents are metformin and DPP-IV inhibitors (eg, sitagliptin, vildagliptin, alogliptin, and saxagliptin).

Suitable anti-obesity agents include 11β-hydroxysteroid dehydrogenase 1 (11β-HSD type 1) inhibitor, stearoyl CoA desaturase 1 (SCD-1) inhibitor, MCR-4 agonist, cholecystokinin A (CCK-A) agonist Drugs, monoamine reuptake inhibitors (such as sibutramine), sympathomimetic drugs, β ₃ adrenergic drugs, dopamine agonists (such as bromocriptine), melanocyte stimulating hormone analogs, 5HT2c agonists, melanin-concentrating hormone antagonists, leptin ( OB protein), leptin analogs, leptin agonists, galanin antagonists, lipase inhibitors (such as tetrahydrolipstatin, ie orlistat), appetite suppressants (such as bombesin agonists), neuropeptide Y antagonists (eg, NPY Y5 antagonists) medicine), PYY _-36 (the inclusion of analog), thyroid mimetics (Thyromimetic agent), dehydroepiandrosterone or an analog thereof, glucocorticoid agonists or antagonists, orexin antagonists, glucagon-like peptide 1 agonists, ciliary Neurotrophic factors (such as Axokine ™ available from Regeneron Pharmaceuticals, Inc., Tarrytown, NY, and Procter & Gamble Company, Cincinnati, Ohio), human agouti-related protein (AGRP) inhibitors, ghrelin antagonists, histamine 3 antagonists Or inverse agonists, neuromedin U agonists, MTP / ApoB inhibitors (eg, gastrointestinal selective MTP inhibitors such as dirlotapide) Opioid antagonist, and the like orexin antagonists.

Preferred anti-obesity agents for use in the combination aspect of the present invention include gastrointestinal selective MTP inhibitors (eg, zirlotapide, mitrapatide and immititapide, R56918 (CAS number 403987), and CAS) No. 913541-47-6), CCKa agonist (eg, N-benzyl-2- [4- (1H-indol-3-ylmethyl) described in PCT Publication No. WO2005 / 116034 or US Publication No. 2005-0267100A1) -5-oxo-1-phenyl-4,5-dihydro-2,3,6,10b-tetraaza-benzo [e] azulen-6-yl] -N-isopropyl-acetamide), 5HT2c agonist (e.g. lorcaserin , MCR4 agonists (e.g., compounds described in US6,818,658), lipase inhibitor (e.g., _cetilistat), the _{PYY 3-36} (herein, _{"PYY 3-36"} is pegylated _{PYY 3- 36} (including, for example, those described in US Publication No. 2006/0178501), opioid antagonists (eg, naltrexone), oleoyl-estrone (CAS number 180003-17-2), obiinepide (obinepitide) (TM30338), pramlintide (Symlin®), tesofensin (NS2330), leptin, liraglutide, bromocriptine, orlistat, exenatide (Byetta®), AOD-9604 (CAS number 221231-10-3) And sibutramine and the like. The compounds of the invention and combination therapies are preferably administered in conjunction with exercise and a sensible diet.

  All US patents and publications cited above are hereby incorporated by reference.

Pharmaceutical formulations The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof admixed with at least one pharmaceutically acceptable excipient. The pharmaceutical compositions include compositions in a form adapted for oral, topical, or parenteral use and can be used to treat diabetes and related conditions as described above.

  The pharmaceutical composition can be formulated for administration by any route known in the art, including subcutaneous, inhalation, oral, topical, parenteral and the like. The pharmaceutical composition can be any form known in the art including, but not limited to, tablets, capsules, powders, granules, lozenges, or liquid formulations such as oral or sterile parenteral solutions or suspensions. But you can.

  Tablets and capsules for oral administration can be in unit dosage form and are combined with conventional excipients such as syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone; lactose, sugar, corn starch Contains fillers such as calcium phosphate, sorbitol, glycine; tableting lubricants such as magnesium stearate, talc, polyethylene glycol, silica; disintegrants such as potato starch; or acceptable wetting agents such as sodium lauryl sulfate It's okay. The tablets may be coated according to methods well known in standard pharmaceutical practice.

  Oral liquid formulations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or dry products that are reconstituted with water or other appropriate vehicle before use. It can also take the form of Such liquid preparations contain conventional additives such as suspending agents such as sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, hydrogenated edible fat; lecithin, monooleic acid Emulsifiers such as sorbitan and acacia; non-aqueous vehicles such as tonsil oil and glycerin, propylene glycol and ethyl alcohol (may include edible oils); methyl or propyl p-hydroxybenzoate, sorbic acid and the like Preservatives and, if desired, conventional flavoring or coloring agents may be included.

  For parenteral administration, fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, water being preferred. The compound can be suspended or dissolved in a vehicle or other suitable solvent, depending on the vehicle and concentration used. In preparing solutions, the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing. For convenience, drugs such as local anesthetics, preservatives, buffering agents can be dissolved in the vehicle. To increase stability, the composition can be frozen after filling into the vial and the water removed in vacuo. The lyophilized dry powder can then be sealed in a vial and supplied with an attached vial of water for injection to reform the liquid prior to use. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and that sterilization cannot be accomplished by filtration. The compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is incorporated into the composition to facilitate uniform distribution of the compound.

  The composition may contain, for example, from about 0.1% to about 99% by weight of active material, depending on the method of administration. When the composition comprises dosage units, each unit will contain, for example, from about 0.1 to 900 mg, more typically from 1 mg to 250 mg of active ingredient.

  The compounds of the present invention can be formulated for administration in any convenient manner for use in human or veterinary medicine by analogy with other antidiabetic agents. Such methods are known in the art and are outlined above. For a more detailed discussion on the preparation of such formulations, please turn to Remington's Pharmaceutical Sciences, 21st edition, by the University of the Sciences in Philadelphia.

  The following examples illustrate embodiments of the invention. However, it should be understood that embodiments of the present invention do not limit the details of the examples, and therefore other variations thereof will be known to those skilled in the art or will be apparent in light of this disclosure. I want to be.

(Example)
Unless otherwise specified, the starting materials are generally from Aldrich Chemicals Co. (Milwaukee, Wis.), Lancaster Synthesis, Inc. (Windham, New Hampshire), Acros Organics (Fair Lawn, New Jersey), Maybridge Chemical Company, Ltd. Available from commercial sources such as (Cornwall, UK), Tyger Scientific (Princeton, NJ), AstraZeneca Pharmaceuticals (London, UK), Mallinckrodt Baker (Philipsburg, NJ), EMD (Gibstown, NJ), etc.

General Experimental Procedures For proton analysis, NMR spectra were obtained at room temperature using a Varian Unity ™ 400 (DG400-5 probe) or 500 (DG500-5 probe) (both available from Varian Inc., Palo Alto, Calif.). Recorded at 400 MHz or 500 MHz, respectively. Chemical shifts are expressed in parts per million (δ) based on residual solvent as an internal standard. The peak shape is expressed as follows. That is, s: single line, d: double line, dd: double double line, t: triple line, q: quadruple line, m: multiple line, bs: broad single line, 2s: two single lines .

  Atmospheric pressure chemical ionization mass spectrum (APCI) was measured using a Waters ™ spectrometer (Micromass ZMD, carrier gas: nitrogen) (available from Waters Corp., Milford, Mass., USA) at a flow rate of 0.3 mL / min. And obtained using a 50:50 water / acetonitrile eluent system. Electrospray ionization mass spectra (ES) were obtained using a Waters ™ (Micromass ZQ or ZMD instrument (carrier gas: nitrogen) (Waters Corp., Milford, Mass.) Liquid chromatography mass spectrometer for each solvent. Acquired using a 95: 5 to 0: 100 gradient water acetonitrile solution with 0.01% formic acid, these instruments include 1 mL / min for 3.75 min or 2 mL / min for 1.95 min A Varian Polaris 5 C18-A 20 × 2.0 mm column (Varian Inc., Palo Alto, Calif.) Was used at a flow rate of

  Column chromatography uses either a Flash 40 Biotage ™ column (ISC, Inc., Shelton, Conn.) Or a Biotage ™ SNAP cartridge KPsil or Redisep Rf silica (from Teledyne Isco Inc), using silica gel. And carried out under nitrogen pressure. Preparative HPLC was performed using a Waters FractionLynx system equipped with a photodiode array (Waters 2996) and a mass spectrometer (Waters / Micromass ZQ) detection scheme. Analytical HPLC work was performed using a Waters 2795 Alliance HPLC or Waters ACQUITY UPLC equipped with a photodiode array, single focusing quadrupole mass detection and evaporative light scattering detection scheme.

  Concentration in vacuo refers to evaporating the solvent under reduced pressure using a rotary evaporator.

  Unless otherwise stated, chemical reactions were performed at room temperature (about 23 degrees Celsius). Also, unless otherwise stated, the chemical reactions were carried out in a nitrogen atmosphere.

Pharmacological Data The practice of the invention to treat diseases modulated by agonist activation of the G protein-coupled receptor GPR119 by a compound of the invention can be demonstrated by activity in one or more of the functional assays described below. . The supplier is shown in parentheses.

In-vitro functional assay β-lactamase:
The assay for GPR119 agonists utilizes a cell-based (hGPR119 HEK293-CRE β-lactamase) reporter construct that combines agonist activation of human GPR119 with β-lactamase production by a cyclic AMP response element (CRE). To do. GPR119 activity is measured using CCF4-AM (Live Blaze FRET-B / G Loading kit, Invitrogen catalog number K1027), which is a β-lactamase substrate that enables FRET. Specifically, hGPR119-HEK-CRE-β-lactamase cells (Invitrogen 2.5 × 10 ⁷ / mL) are removed from a liquid nitrogen reservoir and plated medium (Dulbecco's modified Eagle medium high glucose (DMEM, Gibco catalog number 11995). -065) 10% heat inactivated fetal bovine serum (HIFBS, Sigma catalog number F4135), 1 × MEM nonessential amino acids (Gibco catalog number 15630-080), 25 mM HEPES pH 7.0 (Gibco catalog number 15630-080) ), Diluted in 200 nM potassium clavulanate (Sigma catalog number P3494.) Cell plating medium was used to adjust the cell concentration and 50 μL of this cell suspension (12.5 × 10 ⁴ viable cells) Poly-d- In addition to each well of a thin coated black clear bottom 384 well plate (Greiner Bio-One Cat # 781946), it was incubated at 37 ° C. in a humidified environment containing 5% carbon dioxide. The medium was removed and replaced with 40 μL of assay medium (the assay medium is a plating medium that does not contain potassium clavulanate and HIFBS). The cells were incubated for 16 hours at 37 ° C. in a humidified environment containing 5% carbon dioxide (DMSO ≦ 0.5%) and the plates were removed from the incubator and allowed to equilibrate to room temperature for approximately 15 minutes. 10 uL of 6 × CCF4 / AM working dye solution (Live lazer FRET-B / G Loading kit (prepared according to instructions contained in Invitrogen catalog number K1027) was added per well and incubated for 2 hours at room temperature in the dark EnVision Fluorometric Plate Reader (Excitation 405 nm, luminescence) Fluorescence was measured at 460 nm / 535 nm) EC ₅₀ was determined from agonist response curves analyzed with a curve fitting program using a four parameter logistic dose response equation.

cAMP:
GPR119 agonist activity was also determined in a cellular assay utilizing an HTRF (homogeneous time-resolved fluorescence) cAMP detection kit (cAMP dynamic 2 assay kit Cis Bio catalog number 62AM4PEC) that measures cAMP levels in cells. This method is a competitive immunoassay between natural cAMP produced by cells and cAMP labeled with the dye d2. Tracer binding is visualized with Mab anti-cAMP labeled with cryptate. The specific signal (ie energy transfer) is inversely proportional to the concentration of cAMP in the standard or sample.

Specifically, hGPR119 HEK-CRE β-lactamase cells (Invitrogen 2.5 × 10 ⁷ / mL, the same cell line used in the β-lactamase assay described above) were removed from cryopreservation and grown in growth medium (Dulbecco's modified Eagle medium high Glucose (DMEM, Gibco catalog number 119905-065), 1% charcoal dextran treated fetal calf serum (CD serum, HyClone catalog number SH30068.03), 1 × MEM non-essential amino acids (Gibco catalog number 15630-080), and 25 mM Dilute in HEPES pH 7.0 (Gibco catalog number 15630-080)). The cell concentration was adjusted to 1.5 × 10 ⁵ cells / mL and 30 mL of this suspension was added to a T-175 flask and incubated at 37 ° C. in a humidified environment of 5% carbon dioxide. After 16 hours (overnight), cells were removed from the T-175 flask (by tapping the sides of the flask), centrifuged at 800 × g, and then assay medium (1 × HBSS + CaCl ₂ + MgCl ₂ (Gibco catalog number 14025− 092) and 25 mM HEPES pH 7.0 (Gibco catalog number 15630-080)). Adjust the cell concentration to 6.25 × 10 ⁵ cells / mL using assay medium, and add 8 μl (5000 cells) of this cell suspension to each of the Greiner 384 well white small volume assay plates (VWR catalog number 82051-458). Added to wells.

  Different concentrations of each compound to be tested were diluted in assay buffer containing 3-isobutyl-1-methylxanthine (IBMX, Sigma catalog number I5879) and added to the assay plate wells in a 2 μL volume (final IBMX concentration). Was 400 μM, and the final DMSO concentration was 0.58%). Following a 30 minute incubation at room temperature, 5 μL of labeled d2cAMP and 5 μL of anti-cAMP antibody (both diluted 1:20 in cell lysis buffer, as described in the manufacturer's assay protocol) were added to the assay plate. Added to each well. Plates were then incubated at room temperature and after 60 minutes, changes in the HTRF signal were read on an Envision 2104 multilabel plate reader using excitation wavelength 330 nm, emission wavelengths 615 nm and 665 nm. From the agonist response curve, the raw data was converted to nM cAMP by interpolation from a cAMP calibration curve (as described in the manufacturer's assay protocol) and analyzed with a curve fitting program using a four parameter logistic dose response equation. EC50 was determined.

  It has been observed that the cAMP reaction due to activation of GPR119 can also occur in cells other than the specific cell line used here.

β-arrestin:
GPR119 agonist activity was also determined in cell assays utilizing the DiscoverX PathHunter β-arrestin cell assay technology and its U2OS hGPR119β-arrestin cell line (DiscoverX catalog number 93-0356C3). In this assay, agonist activation is determined by measuring the agonist-induced interaction between β-arrestin and activated GPR119. A small 42-amino acid enzyme fragment called ProLink was added to the C-terminus of GPR119. Arrestin was fused to a larger enzyme fragment called EA (enzyme acceptor). When GPR119 is activated, arrestin binding is stimulated and the complementation of the two enzyme fragments is derived, resulting in the production of a functional β-galactosidase enzyme that can hydrolyze the substrate and generate a chemiluminescent signal.

Specifically, U2OS hGPR119β-arrestin cells (DiscoverX 1 × 10 ⁷ / mL) were removed from cryopreservation and grown in growth medium (minimum essential medium (MEM, Gibco catalog number 11095-080), 10% heat-inactivated fetal bovine serum ( HIFBS, Sigma catalog number F4135-100), 100 mM sodium pyruvate (Sigma catalog number S8636), 500 μg / mL G418 (Sigma catalog number G8168), and 250 μg / mL hygromycin B (Invitrogen catalog number 10687-010) diluted in. the cell concentration was adjusted to 1.66 × 10 ⁵ cells / mL, was added and the suspension 30mL in T-175 flasks and incubated at 37 ° C. in a humidified environment of 5% CO After 24 hours, cells were removed from the T-175 flask using enzyme-free cell dissociation buffer (Gibco catalog number 13151-014), centrifuged at 800 × g, and then plated medium (Opti-MEM I ( Invitrogen / BRL catalog number 31985-070) and 2% charcoal dextran-treated fetal calf serum (CD serum, HyClone catalog number SH30068.03)). Adjust to ⁵ cells / mL and add 10 μL of this cell suspension (2500 cells) to each well of a Greiner 384 well white small volume assay plate (VWR catalog number 82051-458) and add the plate to a humidified 5% carbon dioxide. Incubate at 37 ° C in the environment It was collected.

After 16 hours (overnight), the assay plate was removed from the incubator and tested at various concentrations of each compound (assay buffer (1 × HBSS + CaCl ₂ + MgCl ₂ (Gibco catalog number 14025-092), 20 mM HEPES pH7. 0 (Gibco catalog number 15630-080) and 0.1% BSA (diluted in Sigma catalog number A9576) were added to the assay plate wells in a volume of 2.5 μL (final DMSO concentration of 0.5 After incubation for 90 minutes at 37 ° C. in a humidified environment of 5% carbon dioxide, 7.5 μL of Galacton Starβ-galactosidase substrate (PathHunter Detection Kit (DiscoverRx catalog number 93-0001)) , Prepared as described in the manufacturer's assay protocol) was added to each well of the assay plate, and the plate was incubated at room temperature and after 60 minutes, using an Envision 2104 multilabel plate reader at 0.1 seconds per well. Changes in luminescence were read and EC50s were determined from agonist response curves analyzed with a curve fitting program using a 4-parameter logistic dose response equation.

GPR119 Expression and GPR119 Binding Assay Using BacMam Wild-type human GPR119 by polymerase chain reaction (PCR) (Pfu Turbo Mater Mix, Stratagene, La Jolla, Calif.) Using pIRES-puro-hGPR119 as template and the following primers: (FIG. 1) was amplified.
hGPR119 BamH1, upstream 5'-TAAATTGGATCCACCCATGGAATCATTTTTCTCATTTGGAG-3 '
(Insert a BamHI site at the 5 'end)
hGPR119 EcoRI, downstream 5'-TAAATTGAATTCTTATCAGCCATCAAACTCTGAGC-3 '
(Insert EcoRI site at 3 'end)

  The amplification product was purified (Qiaquick Kit, Qiagen, Valencia, Calif.) And digested with BamH1 and EcoRI (New England BioLabs, Ipswich, Mass.) According to the manufacturer's protocol. The vector pFB-VSVG-CMV-poly (FIG. 2) was digested with BamHI and EcoRI (New England BioLabs, Ipswich, Mass.). Digested DNA was separated by electrophoresis on a 1% agarose gel and fragments were excised from the gel and purified (Qiaquick Kit, Qiagen, Valencia, Calif.). The vector and gene fragment were ligated (Rapid Ligase Kit, Roche, Pleasanton, Calif.) And transformed into OneShot DH5αT1R cells (Invitrogen, Carlsbad, Calif.). Eight ampicillin resistant colonies (“clone 1-8”) were grown for minipreps (Qiagen Miniprep Kit, Qiagen, Valencia, Calif.) And sequenced to confirm identity and correct orientation of insertion.

  The pFB-VSVG-CMV-poly-hGPR119 construct (clone # 1) was transformed into OneShot DH10Bac cells (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Eight positive (ie white) colonies were re-streaked to be identified as “positive” and subsequently grown for bacmid isolation. Recombinant hGPR119 bacmid was isolated by a modified alkaline lysis procedure using a buffer of Qiagen Miniprep Kit (Qiagen, Valencia, CA). Briefly, pelleted cells were dissolved in buffer P1, neutralized in buffer P2, and precipitated with buffer N3. The precipitate was pelleted by centrifugation (17,900 × g for 10 minutes) and the supernatant was combined with isopropanol to precipitate the DNA. The DNA was pelleted by centrifugation (17,900 × g for 30 minutes), washed once with 70% ethanol and resuspended in 50 μL buffer EB (Tris-HCL, pH 8.5). Polymerase chain reaction (PCR) using commercially available primers (M13F, M13R, Invitrogen, Carlsbad, CA) was used to confirm the presence of the hGPR119 insert in the bacmid.

Production of hGPR119 Recombinant Baculovirus Generation of P0 Virus Stock Suspension-adapted Sf9 cells grown in Sf900II medium (Invitrogen, Carlsbad, Calif.) were subjected to the manufacturer's protocol (Cellfectin, Invitrogen, Calif.). 10 μL of hGPR119 bacmid DNA was transfected according to Carlsbad). After 5 days of incubation, conditioned medium (ie, “P0” virus stock) was centrifuged and filtered through a 0.22 μm filter (Steriflip, Millipore, Billerica, Mass.).

Preparation of Frozen Virus (BIIC) Stocks In preparation for long-term virus storage and production (ie, “P1”) virus stock production, frozen BIIC (insect cells infected with baculovirus) stocks were prepared as follows. Suspension adapted Sf9 cells were grown in Sf900II medium (Invitrogen, Carlsbad, Calif.) And infected with hGPR119 P0 virus stock. After growing for 24 hours, the infected cells are gently centrifuged (approximately 100 × g) and resuspended in freezing medium (Sf900II medium containing 10% DMSO, 1% albumin) to a final density. Was 1 × 10 ⁷ cells / mL and frozen in aliquots of 1 mL according to standard freezing protocols.

Preparation of working (“P1”) virus stock Suspension-adapted Sf9 cells grown in Sf900II medium (Invitrogen, Carlsbad, Calif.) Were infected with a 1: 100 dilution of thawed hGPR119 BIIC stock. Incubated for several days (27 ° C. with shaking). When cell viability reached 70%, conditioned media was collected by centrifugation and virus titer determined by ELISA (BaculoElisa Kit, Clontech, Mountain View, CA).

Overexpression of hGPR119 in HEK 293FT cells adapted to suspension In shake flasks, grow HEK293FT cells (Invitrogen, Carlsbad, CA) in 293 Freestyle medium (Invitrogen) supplemented with 50 μg / mL neomycin and 10 mM HEPES. (37C, 8% carbon dioxide, shaking). Cells are gently centrifuged (approximately 500 × g, 10 minutes) and the pellet is resuspended in a mixture of Dulbecco's PBS (Mg ++ / − Ca ++ no) and P1 virus supplemented with 18% fetal calf serum (Sigma Aldrich). The multiplicity of infection (MOI) was 10 and the final cell density was 1.3 × 10 ⁶ / mL (total volume 2.5 liters). Cells were transferred to 5 liters of Wave Bioreactor Wavebag (Wave Technologies, Mass.) And incubated for 4 hours at 27 ° C (17 rocks / min, platform angle 7 °), at the end of the incubation period 30 mM butyric acid An equal volume (2.5 liters) of 293 Freestyle medium supplemented with sodium (Sigma Aldrich) was added (final concentration = 15 mM) and the cells were grown for 20 hours (37 ° C., 8% CO 2 [0.2 liters / min}). , 25 rocks / minute, platform angle 7 °). Cells were collected by centrifugation (3,000 × g, 10 minutes), washed once with DPBS (Ca ++ / Mg ++ not), 0.25 M sucrose, 25 mM HEPES, 0.5 mM EDTA, pH 7.4. And frozen at −80 ° C.

Membrane preparation for radioligand binding assay Frozen cells were thawed on ice and centrifuged at 700 × g (1400 rpm) for 10 minutes at 4 ° C. The cell pellet was resuspended in 20 mL phosphate buffer solution and centrifuged at 1400 rpm for 10 minutes. The cell pellet is then homogenized buffer (10 mM HEPES (Gibco # 15630), pH 7.5, 1 mM EDTA (BioSolutions, # BIO260-15), 1 mM EGTA (Sigma, # E-4378), 0.01 mg / mL Benzamidine (Sigma # B6506), 0.01 mg / mL bacitracin (Sigma # B0125), 0.005 mg / mL leupeptin (Sigma # L8511), 0.005 mg / mL aprotinin (Sigma # A1153)) Turbid and incubated on ice for 10 minutes. The cells were then lysed with 15 gentle strokes in a tight fit glass Dounce homogenizer. The homogenate was centrifuged at 1000 × g (2200 rpm) for 10 minutes at 4 ° C. The supernatant was transferred to a new centrifuge tube on ice. The cell pellet was resuspended in homogenization buffer and centrifuged again at 1000 × g (2200 rpm) for 10 minutes at 4 ° C., after which the supernatant was removed and the pellet was resuspended in homogenization buffer. This process third Repeat, then the combined supernatant to a final concentration of 1U / mL and 6mM each added Benzonase (Novagen # 71206) and MgCl ₂ (Fluka # 63020), and incubated on ice for 1 hour. The solution was then centrifuged at 25,000 × g (15000 rpm) for 20 minutes at 4 ° C., the supernatant was discarded, and the pellet was resuspended in fresh homogenization buffer (no Benzonase and MgCl ₂ ). After repeating the centrifugation step at 25,000 × g, the final membrane pellet was resuspended in homogenization buffer and frozen at −80 ° C. The protein concentration was determined using the Pierce BCA protein assay kit (Pierce Reagent A # 23223 and B # 23224).

Synthesis and purification of [ ³ H] -Compound A

化合物Ａ（４−（１−（４−（メチルスルホニル）フェニル）−３ａ，７ａ−ジヒドロ−１Ｈ−ピラゾロ［３，４−ｄ］ピリミジン−４−イルオキシ）ピペリジン−１−カルボン酸イソプロピル、上で示したとおり）（４ｍｇ、０．００９ｍｍｏｌ）を０．５ｍＬのジクロロメタンに溶解させ、得られる溶液を（１，５−シクロオクタジエン）（ピリジン）（トリシクロヘキシルホスフィン）−イリジウム（Ｉ）ヘキサフルオロホスフェート（Ｊ．Ｏｒｇａｎｏｍｅｔａｌ．Ｃｈｅｍ．１９７９、１６８、１８３）（５ｍｇ、０．００６ｍｍｏｌ）で処理した。反応容器を密封し、溶液をトリチウムガス雰囲気中で１７時間撹拌した。減圧下で反応溶媒を除去し、得られる残渣をエタノールに溶解させた。未精製の［^３Ｈ］−化合物Ａの精製を、以下の条件を使用する分取ＨＰＬＣによって実施した。
カラム：Ａｔｌａｎｔｉｓ、４．６×１５０ｍｍ、５μｍ
移動相Ａ：水／アセトニトリル／ギ酸（９８／２／０．１）
移動相Ｂ：アセトニトリル
勾配： 時間 ％Ｂ
０．００ ３０．０
１．００ ３０．０
１３．００ ８０．０
分析時間：１６分
ポストタイム：５分
流量：１．５ｍＬ／分
注入体積：２０〜５０μＬ
注入溶媒：ＤＭＳＯ
検出：２１０ｎｍおよび２４５ｎｍでのＵＶ

Compound A (4- (1- (4- (methylsulfonyl) phenyl) -3a, 7a-dihydro-1H-pyrazolo [3,4-d] pyrimidin-4-yloxy) piperidine-1-carboxylate, on (As indicated) (4 mg, 0.009 mmol) was dissolved in 0.5 mL dichloromethane and the resulting solution was (1,5-cyclooctadiene) (pyridine) (tricyclohexylphosphine) -iridium (I) hexafluorophosphate. Treated with (J. Organometal. Chem. 1979, 168, 183) (5 mg, 0.006 mmol). The reaction vessel was sealed and the solution was stirred in a tritium gas atmosphere for 17 hours. The reaction solvent was removed under reduced pressure, and the resulting residue was dissolved in ethanol. Purification of the crude [ ³ H] -Compound A was performed by preparative HPLC using the following conditions.
Column: Atlantis, 4.6 × 150 mm, 5 μm
Mobile phase A: water / acetonitrile / formic acid (98/2 / 0.1)
Mobile phase B: acetonitrile gradient: time% B
0.00 30.0
1.00 30.0
13.00 80.0
Analysis time: 16 minutes Post time: 5 minutes Flow rate: 1.5 mL / min Injection volume: 20-50 μL
Injection solvent: DMSO
Detection: UV at 210 nm and 245 nm

The specific activity of purified [ ³ H] -Compound A was determined to be 70 Ci / mmol by mass spectrometry.

Alternatively, binding assays can be performed using [ ³ H] -Compound B.

Synthesis and purification of [ ³ H] -Compound B

化合物Ｂ（４−（１−（４−（メチルスルホニル）フェニル）−１Ｈ−ピラゾロ［３，４−ｄ］ピリミジン−４−イルオキシ）ピペリジン−１−カルボン酸ｔｅｒｔ−ブチル、上に示すとおり）（５ｍｇ、１０．６μｍｏｌ）を１．０ｍＬのジクロロメタンに溶解させ、得られる溶液をＣｒａｂｔｒｅｅ触媒（５ｍｇ、６．２μｍｏｌ）で処理した。反応容器を密閉し、溶液をトリチウムガス雰囲気中で１７時間撹拌した。減圧下で反応溶媒を除去し、得られる残渣をエタノールに溶解させた。未精製［^３Ｈ］−化合物Ｂの精製を７０％ヘキサン／３０％酢酸エチルを溶離液とするシリカゲルフラッシュカラムクロマトグラフィー、続いて６０％石油エーテル／４０％酢酸エチルを溶離液とするシリカゲルフラッシュカラムクロマトグラフィーによって行った。

Compound B (tert-butyl 4- (1- (4- (methylsulfonyl) phenyl) -1H-pyrazolo [3,4-d] pyrimidin-4-yloxy) piperidine-1-carboxylate, as indicated above) ( 5 mg, 10.6 μmol) was dissolved in 1.0 mL of dichloromethane and the resulting solution was treated with Crabtree catalyst (5 mg, 6.2 μmol). The reaction vessel was sealed and the solution was stirred in a tritium gas atmosphere for 17 hours. The reaction solvent was removed under reduced pressure, and the resulting residue was dissolved in ethanol. Purification of crude [ ³ H] -Compound B using silica gel flash column chromatography with 70% hexane / 30% ethyl acetate as eluent, followed by silica gel flash column with 60% petroleum ether / 40% ethyl acetate as eluent Performed by chromatography.

The specific activity of purified [ ³ H] -Compound B was determined to be 57.8 Ci / mmol by mass spectrometry.

GPR119 radioligand binding assay Test compounds were serially diluted in 100% DMSO (JT Baker # 922401). 2 μL of each dilution was added to the appropriate wells of a 96 well plate (in triplicate concentrations). Unlabeled Compound A (or Compound B) was used at a final concentration of 10 μM to measure nonspecific binding.

[ ³ H] -Compound A (or [ ³ H] -Compound B) is bound to binding buffer (50 mM Tris-HCl, pH 7.5 (Sigma # T7443), 10 mM MgCl ₂ (Fluka 63020), 1 mM EDTA ( BioSolutions # BIO260-15), 0.15% bovine serum albumin (Sigma # A7511), 0.01 mg / mL benzamidine (Sigma # B6506), 0.01 mg / mL bacitracin (Sigma # B0125), 0.005 mg Dilute in / mL leupeptin (Sigma # L8511), 0.005 mg / mL aprotinin (Sigma # A1153) to a concentration of 60 nM and add 100 μL to all wells of a 96-well plate (Nalge Nunc # 267245) The added.

  Membranes expressing GPR119 were thawed and diluted in binding buffer to a final concentration of 20 μg / 100 μL per well, and 100 μL of diluted membrane was added to each well of a 96 well plate.

  The plate was incubated for 60 minutes at room temperature (about 25 ° C.) with shaking. The assay was terminated by vacuum filtration on GF / C filter plates (Packard # 600005174) presoaked in 0.3% polyethyleneamine using a Packard harvester. The filter was then washed 6 times using wash buffer (50 mM Tris-HCl, pH 7.5, kept at 4 ° C.). The filter plate was then air dried overnight at room temperature. 30 μl of scintillation fluid (Ready Safe, Beckman Coulter # 141349) was added to each well, the plates were sealed, and the radioactivity associated with each filter was measured using a Wallac Trilux MicroBeta plate-based scintillation counter.

^[3 H] - Compound A (or ^[3 H] - Compound B) Kd of was obtained by carrying out the saturation binding experiments with data analysis by non-linear regression to fit the one-site hyperbola (Graph Pad Prism). IC ₅₀ was determined from competition curves analyzed using a proprietary curve fitting program (SIGHTS) and a four parameter logistic dose response equation. Ki values were calculated from IC ₅₀ values using the Cheng-Prusoff equation.

  The following results were obtained for the β-lactamase and β-arrestin functional assays.

  The following results were obtained for the cAMP assay and the binding assay.

Preparation of starting materials Preparative Example 1: Isomers (4 and 5) of tert-butyl 3-fluoro-4-hydroxypiperidine-1-carboxylate. Details of the experiment are detailed in Scheme A below.

Step A. 4-[(Trimethylsilyl) oxy] -3,6-dihydropyridine-1 (2H) -carboxylate tert-butyl (2)

Ｎ−ｔｅｒｔ−ブトキシカルボニル−４−ピペリドン（３０．０ｇ、０．１５ｍｏｌ）を無水Ｎ，Ｎ−ジメチルホルムアミド（３００ｍＬ）に溶かした室温の溶液に、塩化トリメチルシリル（２２．９ｍＬ、０．１８ｍｏｌ）およびトリエチルアミン（５０．４ｍＬ、０．３６ｍｏｌ）を滴下漏斗で連続的に加えた。得られる溶液を８０℃で終夜加熱し、次いで室温に冷却した。反応混合物を水およびヘプタンで希釈した。層を分離し、水層をヘプタンで抽出した。ヘプタン層を合わせて水およびブラインで順次洗浄し、次いで硫酸マグネシウムで乾燥させた。混合物を濾過し、減圧下で濾液を濃縮して、粗生成物を黄色の油状物として得た。油状物を９０：１０のヘプタン／酢酸エチル中でシリカゲル充填物に通すことにより精製して、表題化合物を無色の油状物（３３．６ｇ、８２％）として得た。¹H NMR (400 MHz, 重水素化クロロホルム) δ 4.78 (br s, 1H), 3.86 (br s, 2H), 3.51
(t, 2H), 2.09 (br s, 2H), 1.45 (s, 9H), 0.18 (s, 9H).

To a room temperature solution of N-tert-butoxycarbonyl-4-piperidone (30.0 g, 0.15 mol) in anhydrous N, N-dimethylformamide (300 mL) was added trimethylsilyl chloride (22.9 mL, 0.18 mol) and Triethylamine (50.4 mL, 0.36 mol) was added continuously via a dropping funnel. The resulting solution was heated at 80 ° C. overnight and then cooled to room temperature. The reaction mixture was diluted with water and heptane. The layers were separated and the aqueous layer was extracted with heptane. The heptane layers were combined and washed sequentially with water and brine and then dried over magnesium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure to give the crude product as a yellow oil. The oil was purified by passing through a plug of silica gel in 90:10 heptane / ethyl acetate to give the title compound as a colorless oil (33.6 g, 82%). ¹ H NMR (400 MHz, deuterated chloroform) δ 4.78 (br s, 1H), 3.86 (br s, 2H), 3.51
(t, 2H), 2.09 (br s, 2H), 1.45 (s, 9H), 0.18 (s, 9H).

Step B. Tert-Butyl 3-fluoro-4-oxopiperidine-1-carboxylate (3)

４−［（トリメチルシリル）オキシ］−３，６−ジヒドロピリジン−１（２Ｈ）−カルボン酸ｔｅｒｔ−ブチル（２８．８ｇ、０．１１ｍｏｌ）をアセトニトリル（３００ｍＬ）に溶かした室温の撹拌した溶液に、Ｓｅｌｅｃｔｆｌｕｏｒ（商標）（４１．４ｇ、０．１２ｍｏｌ）を加えた。得られる淡黄色の懸濁液を室温で１．５時間撹拌した。飽和炭酸水素ナトリウム水溶液（３００ｍＬ）および酢酸エチル（３００ｍＬ）を加え、層を分離した。水層を酢酸エチルで２回抽出し、すべての有機層を合わせ、飽和炭酸水素ナトリウム水溶液およびブラインで順次洗浄し、次いで硫酸マグネシウムで乾燥させた。混合物を濾過し、減圧下で濾液を濃縮して、粗生成物を淡黄色の油状物として得た。この材料を、ヘプタン／酢酸エチル勾配（２：１〜１：１）を用いたシリカゲルでのカラムクロマトグラフィーを繰り返すことにより精製すると、表題化合物が白色の固体（１５．５ｇ、６７％）として得られた。¹H NMR (400 MHz, 重水素化クロロホルム): δ 4.88 (dd, 0.5 H), 4.77 (dd, 0.5H), 4.47
(br s, 1H), 4.17 (ddd, 1H), 3.25 (br s, 1H), 3.23 (ddd, 1H), 2.58 (m, 1H), 2.51
(m, 1H), 1.49 (s, 9H).

To a stirred solution at room temperature of tert-butyl 4-[(trimethylsilyl) oxy] -3,6-dihydropyridine-1 (2H) -carboxylate (28.8 g, 0.11 mol) in acetonitrile (300 mL) was added SelectFluor. (Trademark) (41.4 g, 0.12 mol) was added. The resulting pale yellow suspension was stirred at room temperature for 1.5 hours. Saturated aqueous sodium bicarbonate (300 mL) and ethyl acetate (300 mL) were added and the layers were separated. The aqueous layer was extracted twice with ethyl acetate and all organic layers were combined, washed sequentially with saturated aqueous sodium bicarbonate and brine, then dried over magnesium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure to give the crude product as a pale yellow oil. This material is purified by repeated column chromatography on silica gel using a heptane / ethyl acetate gradient (2: 1 to 1: 1) to give the title compound as a white solid (15.5 g, 67%). It was. ¹ H NMR (400 MHz, deuterated chloroform): δ 4.88 (dd, 0.5 H), 4.77 (dd, 0.5H), 4.47
(br s, 1H), 4.17 (ddd, 1H), 3.25 (br s, 1H), 3.23 (ddd, 1H), 2.58 (m, 1H), 2.51
(m, 1H), 1.49 (s, 9H).

  Alternatively, Step B can be performed as follows by isolating the ketone hydrate.

To a stirred solution at room temperature of tert-butyl 4-[(trimethylsilyl) oxy] -3,6-dihydropyridine-1 (2H) -carboxylate (41.3 g, 0.15 mol) in acetonitrile (500 mL) was added SelectFluor. (Trademark) (56.9 g, 0.16 mol) was added. The resulting pale yellow suspension was stirred at room temperature for 4 hours and 10 minutes. Saturated aqueous sodium bicarbonate and ethyl acetate were added and the layers were separated. The aqueous layer was extracted twice with ethyl acetate and all organic layers were combined, washed sequentially with saturated aqueous sodium bicarbonate and brine, then dried over magnesium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure to give tert-butyl crude 3-fluoro-4-oxopiperidine-1-carboxylate as a white solid. Tert-butyl crude 3-fluoro-4-oxopiperidine-1-carboxylate was suspended in THF (120 mL) and water (120 mL) was added. The resulting solution was stirred at room temperature for 5.5 hours and then concentrated under reduced pressure. The residue was dried in high vacuum, transferred to an Erlenmeyer flask and suspended in dichloromethane (250 mL). The resulting suspension was stirred for 5 minutes and the solid was collected by filtration using a sintered glass funnel. The resulting filter cake was washed thoroughly with dichloromethane (200 mL), a 1: 1 mixture of dichloromethane (200 mL) and heptane (100 mL). The solid was then dried in high vacuum to give tert-butyl 3-fluoro-4,4-dihydroxypiperidine-1-carboxylate (26.4 g). 1H NMR (500 MHz, deuterated dimethyl sulfoxide) δ 1.38 (s, 9 H), 1.49-1.52 (m, 1H), 1.63-1.68 (m, 1 H), 2.82 -3.20
(m, 2 H) 3.75 (br, 1 H), 3.97 (br, 1 H), 4.12 (d, J = 45, 1 H), 5.92 (s, 1 H),
5.97 (s, 1 H).

Step C. Isomer (4 and 5) (racemic) of tert-butyl (R ^* )-3- (S) -fluoro-4- (R) -hydroxypiperidine-1-carboxylate

３−フルオロ−４−オキソピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（１５．５ｇ、７１．３ｍｍｏｌ）をメタノール（１５０ｍＬ）に溶かした０℃の溶液に、水素化ホウ素ナトリウム（３．５１ｇ、９３．７ｍｍｏｌ）を加えた。得られる混合物を０℃で２時間撹拌し、次いで室温に温めた。飽和塩化アンモニウム水溶液（２００ｍＬ）を加え、混合物を酢酸エチルで３回抽出した。抽出物を合わせてブラインで洗浄し、硫酸マグネシウムで乾燥させた。混合物を濾過し、減圧下で濾液を濃縮して、粗生成物混合物を得、これを、ヘプタン−酢酸エチル（３：２〜１：１）を溶離液とするシリカゲルでのカラムクロマトグラフィーによって精製して、第１の溶離生成物である（３，４−トランス）−３−フルオロ−４−ヒドロキシピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（３．８１ｇ、２４％）を淡黄色の油状物として得たが、油状物は静置すると凝固して白色の固体になった。1H NMR (400 MHz, 重水素化クロロホルム) δ 4.35 (ddd, 0.5 H), 4.18 (ddd, 0.5 H), 4.15 (br s, 1H), 3.89-3.74
(m, 2H), 2.97 (br s, 1H), 2.93 (ddd, 1H), 2.47 (s, 1H), 2.05-1.92 (m, 1H),
1.58-1.46 (m, 1H), 1.44 (s, 9H).

To a solution of tert-butyl 3-fluoro-4-oxopiperidine-1-carboxylate (15.5 g, 71.3 mmol) in methanol (150 mL) at 0 ° C. was added sodium borohydride (3.51 g, 93. 7 mmol) was added. The resulting mixture was stirred at 0 ° C. for 2 hours and then warmed to room temperature. Saturated aqueous ammonium chloride solution (200 mL) was added and the mixture was extracted three times with ethyl acetate. The extracts were combined, washed with brine and dried over magnesium sulfate. The mixture is filtered and the filtrate is concentrated under reduced pressure to give a crude product mixture which is purified by column chromatography on silica gel eluting with heptane-ethyl acetate (3: 2 to 1: 1). The first eluting product tert-butyl (3,4-trans) -3-fluoro-4-hydroxypiperidine-1-carboxylate (3.81 g, 24%) as a pale yellow oil However, the oil solidified to a white solid upon standing. 1H NMR (400 MHz, deuterated chloroform) δ 4.35 (ddd, 0.5 H), 4.18 (ddd, 0.5 H), 4.15 (br s, 1H), 3.89-3.74
(m, 2H), 2.97 (br s, 1H), 2.93 (ddd, 1H), 2.47 (s, 1H), 2.05-1.92 (m, 1H),
1.58-1.46 (m, 1H), 1.44 (s, 9H).

The second eluting compound, tert-butyl (3,4-cis) -3-fluoro-4-hydroxy-piperidine-1-carboxylate (10.57 g, 68%) was then isolated as a white solid. . 1H NMR (400 MHz, deuterated chloroform) δ 4.69-4.65 (m, 0.5H), 4.53-4.49 (m, 0.5H), 3.92-3.86 (m, 2H),
3.69 (br s, 1H), 3.39 (br s, 1H), 3.16 (br s, 1H), 2.13 (s, 1H), 1.88-1.73
(m, 2H), 1.44 (s, 9H).

  Alternatively, Step C can be performed as follows starting from the hydrate tert-butyl 3-fluoro-4,4-dihydroxypiperidine-1-carboxylate (Step B).

  To a stirred solution at −35 ° C. of tert-butyl 3-fluoro-4,4-dihydroxypiperidine-1-carboxylate (20.0 g, 85 mmol) in tetrahydrofuran (500 mL) was added a tetrahydrofuran solution of L-selectride (170 mL). 1M, 170 mmol) was added dropwise over 30 minutes. The reaction mixture was warmed to 0 ° C. over 1.5 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride (150 mL) and stirred vigorously for 15 minutes. A pH 7 phosphate buffer solution (150 mL) was added to the 0 ° C. mixture, and then a 35% aqueous hydrogen peroxide solution (150 mL) was added dropwise. The resulting mixture was stirred for 30 minutes and diluted with ethyl acetate. The organic layer is separated and sequentially with water, saturated aqueous sodium thiosulfate, and brine. The organic layer is then dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated under reduced pressure to give a crude product mixture which is eluted with heptane-ethyl acetate (10-60% gradient). To obtain tert-butyl (3,4-cis) -3-fluoro-4-hydroxypiperidine-1-carboxylate (13.9 g) by column chromatography on silica gel [combiflash ISCO 330 g column]. It was.

Step D. Enantiomer of tert-butyl (3,4-cis) -3-fluoro-4-hydroxy-piperidine-1-carboxylate Racemic (3,4-cis) -3-fluoro-4-hydroxy-piperidine- 1 g sample of tert-butyl 1-carboxylate was collected using a Chiralpak AD-H column (10 × 250 mm) using a mobile phase of 90:10 carbon dioxide and ethanol at a flow rate of 10 mL / min. Purified to its enantiomer by high pressure liquid chromatography. The wavelength for monitoring the separation was 210 nm. By use of analytical high pressure chromatography using a Chiralpak AD-H (4.6 mm × 25 cm) column, each with a constant composition mobile phase consisting of 90:10 carbon dioxide and ethanol at a flow rate of 2.5 mL / min, Purity analysis values for each enantiomer were determined. The wavelength for monitoring the peak was 210 nm. The following two isomers were obtained.
Tert-Butyl (3S, 4R) -3-fluoro-4-hydroxypiperidine-1-carboxylate, enantiomer 1 (363 mg): R _t = 2.67 min (100% ee) (optical rotation in dichloromethane = + 21 .2 degrees) and

  The absolute stereochemistry of the tert-butyl isomer of (3,4-cis) -3-fluoro-4-hydroxy-piperidine-1-carboxylic acid was determined using 5- (6- ( (3S, 4R) -3-fluoropiperidin-4-yloxy) -5-methylpyrimidin-4-yl) -1-methyl-1,4,5,6-tetrahydropyrrolo [3,4-c] pyrazole ( 1S)-(+)-camphor sulfonate (determined by analogy to the preparation of the racemate below).

５−（６−｛［（３，４−シス）−３−フルオロピペリジン−４−イル］オキシ｝−５−メチルピリミジン−４−イル）−１−メチル−１，４，５，６−テトラヒドロピロロ［３，４−ｃ］ピラゾール（ラセミ体）の調製
ａ．５−（６−クロロ−５−メチルピリミジン−４−イル）−１−メチル−１，４，５，６−テトラヒドロピロロ［３，４−ｃ］ピラゾールの調製

5- (6-{[(3,4-cis) -3-fluoropiperidin-4-yl] oxy} -5-methylpyrimidin-4-yl) -1-methyl-1,4,5,6-tetrahydro Preparation of pyrrolo [3,4-c] pyrazole (racemate) a. Preparation of 5- (6-chloro-5-methylpyrimidin-4-yl) -1-methyl-1,4,5,6-tetrahydropyrrolo [3,4-c] pyrazole

１−メチル−１，４，５，６−テトラヒドロピロロ［３，４−ｃ］ピラゾール二塩酸塩（２．００ｇ、１０．２ｍｍｏｌ）および４，６−ジクロロ−５−メチルピリミジン（１．６６ｇ、１０．２ｍｍｏｌ）を室温でテトラヒドロフラン（５１ｍＬ）に懸濁させた。これにトリエチルアミン（４．４１ｍＬ、３１．６ｍｍｏｌ）を加えると、混合物に濁りが生じ、フラスコ壁面に褐色の固体が固着した。この混合物を室温で４時間撹拌し、次いでさらに１９時間５０℃に加熱した。反応混合物を室温に冷却し、水（１００ｍＬ）で希釈した。この混合物を酢酸エチル（３×１００ｍＬ）で抽出した。有機抽出物をプールし、ブラインで洗浄し、硫酸ナトリウムで乾燥させ、濾過した。真空中で濾液を減らして乾燥させ、表題化合物を淡褐色の固体（１．９５ｇ、７８％）として得、これをそれ以上精製せずに次のステップで使用した。
¹H NMR
(500 MHz, 重水素化クロロホルム) δ 2.54
(s, 3 H) 3.88 (s, 3 H) 4.90 (app. d, J=3.66 Hz, 4 H) 7.28 (s, 1 H) 8.29 (s, 1
H).

1-methyl-1,4,5,6-tetrahydropyrrolo [3,4-c] pyrazole dihydrochloride (2.00 g, 10.2 mmol) and 4,6-dichloro-5-methylpyrimidine (1.66 g, 10.2 mmol) was suspended in tetrahydrofuran (51 mL) at room temperature. When triethylamine (4.41 mL, 31.6 mmol) was added thereto, the mixture became cloudy, and a brown solid adhered to the flask wall. The mixture was stirred at room temperature for 4 hours and then heated to 50 ° C. for an additional 19 hours. The reaction mixture was cooled to room temperature and diluted with water (100 mL). This mixture was extracted with ethyl acetate (3 × 100 mL). The organic extracts were pooled, washed with brine, dried over sodium sulfate and filtered. The filtrate was reduced in vacuo and dried to give the title compound as a light brown solid (1.95 g, 78%), which was used in the next step without further purification.
¹ H NMR
(500 MHz, deuterated chloroform) δ 2.54
(s, 3 H) 3.88 (s, 3 H) 4.90 (app.d, J = 3.66 Hz, 4 H) 7.28 (s, 1 H) 8.29 (s, 1
H).

b. (3,4-cis) -3-fluoro-4-{[5-methyl-6- (1-methyl-4,6-dihydropyrrolo [3,4-c] pyrazol-5 (1H) -yl) pyrimidine Preparation of tert-butyl (racemate) of -4-yl] oxy} piperidine-1-carboxylate

（３，４−シス）−３−フルオロ−４−ヒドロキシピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（１．６７ｇ、７．６２ｍｍｏｌ）と上で調製した５−（６−クロロ−５−メチルピリミジン−４−イル）−１−メチル−１，４，５，６−テトラヒドロピロロ［３，４−ｃ］ピラゾール（９００ｍｇ、３．６０ｍｍｏｌ）の混合物を１，４−ジオキサン（２０ｍＬ）に溶解させ、１０５℃に加熱した。１０分間加熱した後、すべての材料が溶液になったので、混合物にナトリウムビス（トリメチルシリル）アミド（４．３ｍＬ、４．３ｍｍｏｌ、１Ｍトルエン溶液）を速やかに加えると、濁った黄色の混合物となり、次いでこれを１０５℃で２時間撹拌した。次いで反応液を室温に冷却し、水と飽和炭酸水素ナトリウム水溶液の等体積混合物を加えて失活させた。混合物を酢酸エチル（３×１５ｍＬ）で抽出した。有機抽出物を合わせてブラインで洗浄し、硫酸ナトリウムで乾燥させ、濾過した。濾液を真空中で濃縮して黄色の残渣を得、これを、６０〜１００％の酢酸エチルヘプタン溶液を溶離液とするシリカゲルでのカラムクロマトグラフィーによって精製した。表題化合物と出発材料の５−（６−クロロ−５−メチルピリミジン−４−イル）−１−メチル−１，４，５，６−テトラヒドロピロロ［３，４−ｃ］ピラゾールの混合物を白色の固体（１．２０ｇ）として単離し、それ以上精製せずに後続の反応で使用した。

Tert-Butyl (3,4-cis) -3-fluoro-4-hydroxypiperidine-1-carboxylate (1.67 g, 7.62 mmol) and 5- (6-chloro-5-methylpyrimidine- prepared above 4-yl) -1-methyl-1,4,5,6-tetrahydropyrrolo [3,4-c] pyrazole (900 mg, 3.60 mmol) was dissolved in 1,4-dioxane (20 mL) and 105 Heated to ° C. After heating for 10 minutes, all the material went into solution, so when sodium bis (trimethylsilyl) amide (4.3 mL, 4.3 mmol, 1M toluene solution) was quickly added to the mixture, a cloudy yellow mixture was obtained, This was then stirred at 105 ° C. for 2 hours. The reaction was then cooled to room temperature and quenched by the addition of an equal volume mixture of water and saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate (3 × 15 mL). The combined organic extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give a yellow residue which was purified by column chromatography on silica gel eluting with 60-100% ethyl acetate heptane solution. A mixture of the title compound and the starting material 5- (6-chloro-5-methylpyrimidin-4-yl) -1-methyl-1,4,5,6-tetrahydropyrrolo [3,4-c] pyrazole Isolated as a solid (1.20 g) and used in subsequent reactions without further purification.

A batch of crude (3,4-cis) -3-fluoro-4-{[5-methyl-6- (1-methyl-4,6-dihydropyrrolo [ Tert-Butyl 3,4-c] pyrazol-5 (1H) -yl) pyrimidin-4-yl] oxy} piperidine-1-carboxylate was purified by HPLC. A crude sample (9.5 mg) was dissolved in dimethyl sulfoxide (1 mL) and from 80% water / acetonitrile (0.03% ammonium hydroxide modifier) to 0% water / acetonitrile in 8.5 min. It was then purified by preparative reverse phase HPLC on a Waters XBridge C ₁₈ 19 × 100 mm, 0.005 mm column eluting with 0% water / acetonitrile for 1.5 min linear gradient, flow rate: 25 mL / min. Thus, the title compound (5 mg) was obtained. Analytical LCMS: Retention time 2.81 min (Waters XBridge C ₁₈ 4.6 × 50 mm, 0.005 mm column, linear gradient from 90% water / acetonitrile to 5% water / acetonitrile over 4.0 min, followed by 5% water / acetonitrile for 1 minute, 0.03% ammonium hydroxide modifier, flow rate: 2.0 mL / min), LCMS (ES +) 433.2 (M + 1).

c. 5- (6-{[(3,4-cis) -3-fluoropiperidin-4-yl] oxy} -5-methylpyrimidin-4-yl) -1-methyl-1,4,5,6-tetrahydro Preparation of pyrrolo [3,4-c] pyrazole (racemate)

上で調製した未精製（３，４−シス）−３−フルオロ−４−｛［５−メチル−６−（１−メチル−４，６−ジヒドロピロロ［３，４−ｃ］ピラゾール−５（１Ｈ）−イル）ピリミジン−４−イル］オキシ｝ピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（１．２０ｇ）をジクロロメタン（１２ｍＬ）に溶解させ、この溶液にトリフルオロ酢酸（５ｍＬ）を加えた。反応液を室温で１時間撹拌した。真空下で溶媒を除去し、残渣を水（５０ｍＬ）および１Ｎ塩酸水溶液（１０ｍＬ）に溶解させた。混合物をジクロロメタン（１０×３０ｍＬ）で抽出した。次いで、１Ｎ水酸化ナトリウム水溶液（２０ｍＬ）を加えて水層をｐＨ１２とし、ジクロロメタン（４０ｍＬ）で３回抽出した。有機抽出物を合わせてブラインで洗浄し、硫酸ナトリウムで乾燥させ、濾過した。濾液を減圧下で濃縮して、５−（６−｛［（３，４−シス）−３−フルオロピペリジン−４−イル］オキシ｝−５−メチルピリミジン−４−イル）−１−メチル−１，４，５，６−テトラヒドロピロロ［３，４−ｃ］ピラゾール（０．７２ｇ、２ステップで６０％）を白色の固体として得、これをさらに精製せずに使用した。
¹H NMR
(500 MHz, 重水素化クロロホルム) δ 1.84 -
2.08 (m, 2 H) 2.33 (s, 3 H) 2.69 - 2.84 (m, 1 H) 2.83 - 3.01 (m, 1 H) 3.16 (d,
J=13.66 Hz, 1 H) 3.27 - 3.44 (m, 1 H) 3.86 (s, 3 H) 4.78-4.91 (m, 1 H) 4.86 (d,
J=1.95 Hz, 2 H) 4.88 (d, J=1.95 Hz, 2 H) 5.21 - 5.32 (m, 1 H) 7.26 (s, 1 H)
8.18 (s, 1 H); LCMS (ES+) 333.4 (M+1).
（３Ｒ，４Ｓ）−３−フルオロ−４−ヒドロキシピペリジン−１−カルボン酸ｔｅｒｔ−ブチル、鏡像異性体２（４０３ｍｇ）：Ｒ_ｔ＝２．９９分（８８％ｅｅ）。

The crude (3,4-cis) -3-fluoro-4-{[5-methyl-6- (1-methyl-4,6-dihydropyrrolo [3,4-c] pyrazole-5 ( 1H) -yl) pyrimidin-4-yl] oxy} piperidine-1-carboxylate tert-butyl (1.20 g) was dissolved in dichloromethane (12 mL) and trifluoroacetic acid (5 mL) was added to this solution. The reaction was stirred at room temperature for 1 hour. The solvent was removed under vacuum and the residue was dissolved in water (50 mL) and 1N aqueous hydrochloric acid (10 mL). The mixture was extracted with dichloromethane (10 × 30 mL). Next, 1N aqueous sodium hydroxide solution (20 mL) was added to adjust the aqueous layer to pH 12, and the mixture was extracted 3 times with dichloromethane (40 mL). The combined organic extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 5- (6-{[(3,4-cis) -3-fluoropiperidin-4-yl] oxy} -5-methylpyrimidin-4-yl) -1-methyl- 1,4,5,6-tetrahydropyrrolo [3,4-c] pyrazole (0.72 g, 60% over 2 steps) was obtained as a white solid, which was used without further purification.
¹ H NMR
(500 MHz, deuterated chloroform) δ 1.84-
2.08 (m, 2 H) 2.33 (s, 3 H) 2.69-2.84 (m, 1 H) 2.83-3.01 (m, 1 H) 3.16 (d,
J = 13.66 Hz, 1 H) 3.27-3.44 (m, 1 H) 3.86 (s, 3 H) 4.78-4.91 (m, 1 H) 4.86 (d,
J = 1.95 Hz, 2 H) 4.88 (d, J = 1.95 Hz, 2 H) 5.21-5.32 (m, 1 H) 7.26 (s, 1 H)
8.18 (s, 1 H); LCMS (ES +) 333.4 (M + 1).
Tert-Butyl (3R, 4S) -3-fluoro-4-hydroxypiperidine-1-carboxylate, enantiomer 2 (403 mg): R _t = 2.99 min (88% ee).

Preparation Example 2: 9-Hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate isopropyl (mixture of syn and anti isomers)

Step A of Scheme B. Synthesis of 7-benzyl-3-oxa-7-azabicyclo [3.3.1] nonan-9-one-hydrochloride (2):
Tetrahydro-4H-pyran-4-one 1 (60.0 g, 0.60 mol), benzylamine (63.4 g, 0.60 mol), and glacial acetic acid (35.9 g, 0.60 mol) were added to anhydrous methanol (1. 2 L) was added to a stirred suspension of paraformaldehyde (39.6 g, 1.3 mol) in anhydrous methanol (1.2 L) at 65 ° C. over 75 minutes. A second portion of paraformaldehyde (39.6 g, 1.3 mol) was added and the mixture was stirred at 65 ° C. for 1 hour. The reaction was quenched with water (1.2 L) and 1M aqueous potassium hydroxide (600 mL). The mixture was extracted with ethyl acetate (3 L × 3). The organic layers were combined, dried over sodium sulfate, filtered, and the filtrate was concentrated to dryness in vacuo. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 20: 1 to 2: 1) to give a brown oil. The residue was diluted with 6M anhydrous hydrochloric acid 1,4 dioxane solution (500 mL) and the mixture was stirred for 30 minutes. The solvent was removed in vacuo and acetone (500 mL) was added. The resulting mixture was sonicated for 30 minutes to produce a white precipitate. The mixture was filtered and the solid was washed with acetone and then dried in vacuo to give the desired product as a white solid (21 g, 13%). ¹ H NMR (400 MHz, deuterium oxide)
δ 7.43-7.42 (m, 5H), 4.66 (s, 2H), 3.95-3.90 (m, 4H),
3.54-3.47 (m, 4H); 1.96 (bs, 2H); LCMS (ES +): 232.0 (M + 1).

Step B of Scheme B. Synthesis of 7-benzyl-3-oxa-7-azabicyclo [3.3.1] nonan-9-ol (mixture of syn and anti isomers) (3):
7-Benzyl-3-oxa-7-azabicyclo [3.3.1] nonan-9-one hydrochloride (4.40 g, 16.9 mmol) was suspended in ethanol (40 mL) and anhydrous tetrahydrofuran (40 mL). . The mixture was cooled in an ice bath and sodium borohydride (1.5 g, 37.3 mmol) was added in one portion. The mixture was slowly warmed to room temperature over 4 hours. The reaction was then concentrated in vacuo to remove most of the ethanol and tetrahydrofuran. The mixture was partitioned between methyl tert-butyl ether and 1.0 M aqueous sodium hydroxide. After stirring the solution for 30 minutes, the two layers were separated. The aqueous layer was extracted with methyl tert-butyl ether. The organic extracts were combined, washed with brine and dried over sodium sulfate. The mixture was filtered and the filtrate was concentrated in vacuo to give a clear oil that solidified partially upon standing to an oily white solid (3.71 g, 94%). This mixture of syn and anti 7-benzyl-3-oxa-7-azabicyclo [3.3.1] nonan-9-ol isomers was used in the next step without further purification. LCMS (ES +): 234.1 (M + 1).

Step C of Scheme B. Synthesis of 3-oxa-7-azabicyclo [3.3.1] nonan-9-ol (mixture of syn and anti isomers) (4):
A starting mixture of syn and anti 7-benzyl-3-oxa-7-azabicyclo [3.3.1] nonane-9-ol isomers (3.71 g, 15.9 mmol) was dissolved in ethanol (120 mL), Pd (OH) ₂ (450 mg) was added. The mixture was shaken in 50 psi hydrogen for 2.5 hours on a Parr shaker. The mixture was filtered through Celite® and the collected solid was washed 3 times with methanol. The filtrate was concentrated in vacuo to give an oily solid. This oily solid was dissolved in ethyl acetate and heptane was added. Concentration of the solution in vacuo gave a syn and anti isomer mixture of 3-oxa-7-azabicyclo [3.3.1] nonan-9-ol as a white solid (2.08 g, 91%). . This material was used in the next step without further purification. LCMS (ES +): 144.1 (M + 1).

Step D of Scheme B. Synthesis of 9-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate (mixture of syn and anti isomers) (5):
Syn and anti isomer mixture of 3-oxa-7-azabicyclo [3.3.1] nonane-9-ol (2.08 g, 14.5 mmol) and N, N diisopropylethylamine (2.80 mL, 16.0 mmol) To a 0 ° C. dichloromethane (15 mL) solution was added dropwise isopropyl chloroformate (14.2 mL, 14.2 mmol, 1.0 M toluene solution). The reaction mixture was warmed to room temperature over 14 hours. Subsequently, the reaction liquid was diluted with 1M hydrochloric acid aqueous solution (50 mL), and the aqueous layer was separated. The organic layer was washed sequentially with water (50 mL) and brine (50 mL) and dried over sodium sulfate. The mixture was filtered and the filtrate was concentrated in vacuo to give a colorless oil. This oil was dissolved in ethyl acetate, heptane was added and the mixture was concentrated. The resulting oil was dried in vacuo to give a mixture of syn and anti isomers of isopropyl 9-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate as a clear oil ( 2.74 g, 82%). LCMS (ES +): 230.1 (M + 1).

Step E. Separation of syn and anti isomers of isopropyl 9-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate:
By preparative high pressure liquid chromatography using a Chiralpak AD-H column (21 × 250 mm) with 85:15 carbon dioxide and methanol each at a flow rate of 65 mL / min as the mobile phase, 9-hydroxy-3-oxa- A syn and anti isomer mixture of isopropyl 7-azabicyclo [3.3.1] nonane-7-carboxylate (5.04 g, 35.1 mmol) was isolated. The wavelength for monitoring separation was 210 nm. By analytical high-pressure chromatography using a Chiralpak AD-H (4.6 mm × 25 cm) column with 85:15 carbon dioxide and methanol each at a flow rate of 2.5 mL / min as mobile phases. The purity of each isomer was determined. The wavelength for monitoring the peak was 210 nm. The following two isomers were obtained.
9-Syn-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate isopropyl (6) (1.34 g): a clear oil that solidified on standing. . Retention time (R _t ) = 2.3 min, ¹ H NMR (400 MHz, deuterated-DMSO):
δ 5.12 (d, 1H, J = 2.8Hz), 4.76-4.71 (m, 1H), 4.20 (d, 1H,
J = 13Hz), 4.16 (d, 1H, J = 13Hz), 3.96-3.92 (m, 2H), 3.79 (d, 1H, J = 3Hz), 3.55
(s, 1H), 3.52 (S, 1H), 3.08 (d, 1H, J = 13Hz), 2.98 (d, 1H, J = 13Hz), 1.47 (m, 2H)
1.16 (d, 3H, J = 3Hz), 1.15 (d, 3H, J = 3Hz); LCMS (ES +): 230.2 (M + 1).
Isopropyl 9-anti-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate (7) (1.70 g): amber oil, R _t = 3.08 min. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 5.11 (d, 1H, J = 2.8Hz), 4.74-4.67 (m, 1H), 3.89 (d, 1H, J = 13Hz),
3.84-3.78 (m, 2H, J = 11Hz), 3.80 (d, 1H, J = 6Hz), 3.78 (d, 1H, J = 3Hz), 3.52-
3.47 (m, 2H), 3.35-3.30 (m, 1H), 3.24-3.20 (m, 1H), 1.53 (s, 1H), 1.51 (s,
1H), 1.13 (d, 3H, J = 1Hz), 1.16 (d, 3H, J = 1Hz); LCMS (ES +): 230.2 (M + 1)

Alternatively, Steps A and B of Reaction Scheme A above are combined as described below to produce 7-benzyl-3-oxa-7-azabicyclo [3.3.1] nonan-9-ol (syn and anti-isomerism). Body mixtures) can also be synthesized.
Benzylamine (21.35 g, 199.27 mmol), tetrahydro-4H-pyran-4-one (1) (19.95 g, 199.27 mmol), and acetic acid (11.97 g, 199.27 mmol) in methanol (400 mL) Dissolved in. The mixture was heated to reflux. To the reaction mixture, an aqueous formaldehyde solution (37%, 32.34 g, 398.53 mmol) and methanol (100 mL) were added over 60 minutes while the reaction solution was refluxed. The reaction was cooled to room temperature. Then sodium bicarbonate (16.74 g, 199.27 mmol) was added in small portions. Subsequently, sodium borohydride (7.92 g 209.23 mmol) was added in small portions while keeping the reaction temperature below 25 ° C. The mixture was stirred at ambient temperature for 30 minutes. Celite® (20 g) was added followed by water (100 mL) and 1N aqueous sodium hydroxide (100 mL). After stirring for 1 hour, the mixture was filtered and the filter cake was rinsed sequentially with methanol and water (20 mL each). The filtrate was concentrated in vacuo to remove most of the methanol. The resulting aqueous mixture was extracted with 2-methyltetrahydrofuran (300 mL). The organic phase was washed with brine solution (100 mL), dried over anhydrous magnesium sulfate and concentrated in vacuo to give syn and anti-7-benzyl-3-oxa-7-azabicyclo [3.3.1] nonane- A mixture of 9-ol isomers was obtained as an oil that solidified on standing at room temperature (22.0 g, 47.3%).

Preparation 3: 9-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate tert-butyl (mixture of syn and anti isomers)

３−オキサ−７−アザビシクロ［３．３．１］ノナン−９−オール（シンおよびアンチ異性体の混合物、調製例２ステップＣの生成物）（３．７８ｇ、２６．４ｍｍｏｌ）を水（３０ｍＬ）およびテトラヒドロフラン（３０ｍＬ）に溶かした０℃の溶液に、二炭酸ジ−ｔｅｒｔ−ブチル（５．７６ｇ、２６．４ｍｍｏｌ）のテトラヒドロフラン（２０ｍＬ）溶液を滴下添加した。溶液を徐々に室温に温めながら約１５時間撹拌した。反応液をジクロロメタンおよび水で希釈した。層を分離し、水層をジクロロメタンで抽出した。有機層を合わせ、硫酸ナトリウムで乾燥させた。混合物を濾過し、濾液を減圧下で濃縮すると、表題化合物が透明な油状物（６．５５ｇ）として現れ、これをそれ以上精製せずに使用した。

3-Oxa-7-azabicyclo [3.3.1] nonane-9-ol (mixture of syn and anti isomers, product of Preparation 2, Step C) (3.78 g, 26.4 mmol) in water (30 mL ) And a solution of di-tert-butyl dicarbonate (5.76 g, 26.4 mmol) in tetrahydrofuran (20 mL) was added dropwise to a solution at 0 ° C. dissolved in tetrahydrofuran (30 mL). The solution was stirred for about 15 hours while gradually warming to room temperature. The reaction was diluted with dichloromethane and water. The layers were separated and the aqueous layer was extracted with dichloromethane. The organic layers were combined and dried over sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure to reveal the title compound as a clear oil (6.55 g) that was used without further purification.

Preparation Example 4: Separation of syn and anti isomers of tert-butyl 9-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate

調製例３の９−ヒドロキシ−３−オキサ−７−アザビシクロ［３．３．１］ノナン−７−カルボン酸ｔｅｒｔ−ブチル（５．０４ｇ、３５．１ｍｍｏｌ）のシンおよびアンチ異性体の混合物を、それぞれ８５：１５の二酸化炭素およびメタノールを移動相とし、流量を６５ｍＬ／分としたＣｈｉｒａｌｐａｋ ＡＤ−Ｈカラム（２１×２５０ｍｍ）を利用する分取高圧液体クロマトグラフィーによって分離した。分離をモニターする波長は２１０ｎｍとした。各異性体の純度分析値は、それぞれ８５：１５の二酸化炭素およびメタノールを移動相とし、流量を２．５ｍＬ／分としたＣｈｉｒａｌｐａｋ ＡＤ−Ｈ（４．６ｍｍ×２５ｃｍ）カラムを使用する分析高速クロマトグラフィーを使用して求めた。ピークをモニターする波長は２１０ｎｍとした。以下の２種の異性体が得られた。
９−アンチ−ヒドロキシ−３−オキサ−７−アザビシクロ［３．３．１］ノナン−７−カルボン酸ｔｅｒｔ−ブチル：（１．３０ｇ、１００％ｄｅ）、静置すると白色の固体に凝固した透明な油状物、保持時間（Ｒ_ｔ）＝３．１５分。1H NMR (400 MHz, 重水素化クロロホルム) δ 1.44 (s, 9 H), 1.66 (d, J=16.79 Hz, 2 H), 1.84 (d, J=2.93 Hz, 1 H),
3.30 - 3.52 (m, 2 H), 3.64 (t, J=11.03 Hz, 2 H), 3.93 - 4.21 (m, 5 H).
９−シン−ヒドロキシ−３−オキサ−７−アザビシクロ［３．３．１］ノナン−７−カルボン酸ｔｅｒｔ−ブチル：（１．６４ｇ、８９％ｄｅ）、静置すると白色の固体に凝固した透明な油状物、Ｒ_ｔ＝３．５５分。1H NMR (400 MHz, 重水素化クロロホルム) δ 1.47 (s, 9 H), 1.64 (d, J=13.47 Hz, 2 H), 2.12 (d, J=3.32 Hz, 1 H),
2.92 - 3.22 (m, 2 H), 3.71 - 3.83 (m, 2 H), 3.99 (d, J=3.32 Hz, 1 H), 4.09 -
4.19 (m, 2 H), 4.32 (d, J=13.66 Hz, 1 H), 4.48 (d, J=13.66 Hz, 1 H).

A mixture of the syn and anti isomers of tert-butyl 9-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate (5.04 g, 35.1 mmol) of Preparation 3 was prepared. Each was separated by preparative high pressure liquid chromatography using a Chiralpak AD-H column (21 × 250 mm) with 85:15 carbon dioxide and methanol as mobile phases and a flow rate of 65 mL / min. The wavelength for monitoring separation was 210 nm. The purity analysis value of each isomer was analytical high-speed chromatography using a Chiralpak AD-H (4.6 mm × 25 cm) column with carbon dioxide and methanol of 85:15 as mobile phases and a flow rate of 2.5 mL / min. Determined using graphy. The wavelength for monitoring the peak was 210 nm. The following two isomers were obtained.
9-anti-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate tert-butyl: (1.30 g, 100% de), clear to solidify on white solid upon standing such oil, retention time _(R t) = 3.15 min. 1H NMR (400 MHz, deuterated chloroform) δ 1.44 (s, 9 H), 1.66 (d, J = 16.79 Hz, 2 H), 1.84 (d, J = 2.93 Hz, 1 H),
3.30-3.52 (m, 2 H), 3.64 (t, J = 11.03 Hz, 2 H), 3.93-4.21 (m, 5 H).
Tert-butyl 9-syn-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate: (1.64 g, 89% de), clear to solidify on white solid upon standing Oil, R _t = 3.55 min. 1H NMR (400 MHz, deuterated chloroform) δ 1.47 (s, 9 H), 1.64 (d, J = 13.47 Hz, 2 H), 2.12 (d, J = 3.32 Hz, 1 H),
2.92-3.22 (m, 2 H), 3.71-3.83 (m, 2 H), 3.99 (d, J = 3.32 Hz, 1 H), 4.09-
4.19 (m, 2 H), 4.32 (d, J = 13.66 Hz, 1 H), 4.48 (d, J = 13.66 Hz, 1 H).

Preparation Example 5: Isopropyl 4-[(6-chloro-pyrimidin-4-yl) oxy] piperidine-1-carboxylate

４−ヒドロキシピペリジン−１−カルボン酸イソプロピル（５５３ｍｇ、２．９５ｍｍｏｌ）を無水テトラヒドロフラン（２０ｍＬ）に溶かした溶液に、カリウムｔｅｒｔ−ブトキシド（０．４５０ｍＬ、４．００ｍｍｏｌ）を０℃で加えた。反応混合物を６５℃で１０分間撹拌した。上記混合物に４，６−ジクロロピリミジン（０．４００ｇ、２．６８ｍｍｏｌ）を加えた。次いで、得られる溶液を６５℃で１時間撹拌した。混合物を周囲温度に冷却し、水（１００ｍＬ）で失活させ、酢酸エチル（１００ｍＬ×３）で抽出した。有機層を合わせてブラインで洗浄し、硫酸ナトリウムで乾燥させ、濾過し、濾液を減圧下で濃縮した。残渣を、シリカゲルでのカラムクロマトグラフィー（石油エーテル：酢酸エチル＝２０：１）によって精製して、生成物を白色の固体（３５０ｍｇ、４４％）として得た。

To a solution of isopropyl 4-hydroxypiperidine-1-carboxylate (553 mg, 2.95 mmol) in anhydrous tetrahydrofuran (20 mL) was added potassium tert-butoxide (0.450 mL, 4.00 mmol) at 0 ° C. The reaction mixture was stirred at 65 ° C. for 10 minutes. To the above mixture was added 4,6-dichloropyrimidine (0.400 g, 2.68 mmol). The resulting solution was then stirred at 65 ° C. for 1 hour. The mixture was cooled to ambient temperature, quenched with water (100 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 20: 1) to give the product as a white solid (350 mg, 44%).

Preparation Example 6: Isopropyl 4-[(6-chloro-5-methylpyrimidin-4-yl) oxy] piperidine-1-carboxylate

４−ヒドロキシピペリジン−１−カルボン酸イソプロピル（４８２ｍｇ、２．６８ｍｍｏｌ）の無水テトラヒドロフラン（１５ｍＬ）溶液に、０℃でカリウムｔｅｒｔ−ブトキシド（０．４１ｇ、３．６ｍｍｏｌ）を加えた。反応混合物を６５℃で１０分間撹拌した。上記混合物に４，６−ジクロロ−５−メチルピリミジン（０．４０ｇ、２．４ｍｍｏｌ）を加えた。次いで、得られる溶液を６５℃で１時間撹拌した。混合物を周囲温度に冷却し、水（１００ｍＬ）で失活させ、酢酸エチル（１００ｍＬ×３）で抽出した。有機抽出物を合わせてブラインで洗浄し、硫酸ナトリウムで乾燥させ、濾過し、減圧下で濾液を濃縮した。残渣をシリカゲルでのカラムクロマトグラフィー（石油エーテル：酢酸エチル＝２０：１）によって精製して、表題化合物を白色の固体（６８０ｍｇ、８０％）として得た。

To a solution of isopropyl 4-hydroxypiperidine-1-carboxylate (482 mg, 2.68 mmol) in anhydrous tetrahydrofuran (15 mL) was added potassium tert-butoxide (0.41 g, 3.6 mmol) at 0 ° C. The reaction mixture was stirred at 65 ° C. for 10 minutes. To the above mixture was added 4,6-dichloro-5-methylpyrimidine (0.40 g, 2.4 mmol). The resulting solution was then stirred at 65 ° C. for 1 hour. The mixture was cooled to ambient temperature, quenched with water (100 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 20: 1) to give the title compound as a white solid (680 mg, 80%).

Preparation Example 7: 1-methylcyclopropyl carbonate 4-nitrophenyl carbonate

Ａ）１−メチルシクロプロパノール
１Ｌのフラスコに、チタンメトキシド（１００ｇ）、シクロヘキサノール（２３２ｇ）、およびトルエン（４６１ｍＬ）を入れた。フラスコにＤｅａｎ−Ｓｔａｒｋトラップおよび冷却器を取り付けた。メタノールが除去されるまで、混合物を１４０℃で加熱した。トルエンを１８０℃で除去した。またトルエンを加え、このプロセスを２回繰り返した。すべてのトルエンを除去した後、フラスコを高真空中で乾燥させた。フラスコにジエチルエーテル（５８０ｍＬ）を加えて、１Ｍのジエチルエーテル溶液を調製した。５Ｌの三口フラスコに、オーバーヘッド撹拌子、不活性ガス導入口、および均圧滴下漏斗を取り付けた。フラスコを窒素ガスでフラッシュし、酢酸メチル（６０．１ｍＬ、７５６ｍｍｏｌ）、チタンシクロヘキシルオキシド（１Ｍエーテル溶液７５．６ｍＬ）、およびジエチルエーテル（１５００ｍＬ）を入れた。反応フラスコを室温の水浴に浸した状態で溶液を撹拌した。滴下漏斗に３Ｍの臭化エチルマグネシウム溶液（５５４ｍＬ、１．６６モル）を入れた。グリニャール試薬を室温で３時間かけて滴下添加した。混合物が淡黄色の溶液になり、次いで徐々に沈殿が生成し、最終的に濃い緑色／褐色／黒色の混合物に変わった。グリニャールを加えたのに続き、さらに１５分間撹拌した後、混合物を、１０％濃縮硫酸を１Ｌの水に混ぜた混合物中に慎重に注いだ。得られる混合物を、すべての固体が溶解するまで撹拌した。水層を分離し、ジエチルエーテル２×５００ｍＬで抽出した。有機抽出物を合わせて水、ブラインで順次洗浄し、炭酸カリウム（５００ｇ）で３０分間乾燥させ、濾過し、濾液を真空中で濃縮して油状物を得た。炭酸水素ナトリウム（２００ｍｇ）を加え、未精製材料を蒸留し、１００℃付近で沸騰する画分を集めて、少量の不純物としてメチルエチルケトンおよび２−ブタノールを伴う表題化合物（２３グラム）を得た。¹H NMR (500 MHz, 重水素化クロロホルム) δ 0.45 (app. t, J=6.59 Hz, 2 H), 0.77
(app. t, J=5.61 Hz, 2 H), 1.46 (s, 3 H).表題化合物の調製は、ＷＯ０９１０５７１７にも記載されている。

A) 1-methylcyclopropanol A 1 L flask was charged with titanium methoxide (100 g), cyclohexanol (232 g), and toluene (461 mL). The flask was equipped with a Dean-Stark trap and a condenser. The mixture was heated at 140 ° C. until the methanol was removed. Toluene was removed at 180 ° C. Toluene was also added and the process was repeated twice. After removing all the toluene, the flask was dried in high vacuum. Diethyl ether (580 mL) was added to the flask to prepare a 1M diethyl ether solution. An overhead stirrer, inert gas inlet, and pressure equalizing dropping funnel were attached to a 5 L three-necked flask. The flask was flushed with nitrogen gas and charged with methyl acetate (60.1 mL, 756 mmol), titanium cyclohexyl oxide (75.6 mL of 1M ether solution), and diethyl ether (1500 mL). The solution was stirred while the reaction flask was immersed in a room temperature water bath. A dropping funnel was charged with 3M ethylmagnesium bromide solution (554 mL, 1.66 mol). Grignard reagent was added dropwise at room temperature over 3 hours. The mixture became a light yellow solution and then gradually a precipitate formed, eventually turning to a dark green / brown / black mixture. Following the addition of Grignard and stirring for an additional 15 minutes, the mixture was carefully poured into a mixture of 10% concentrated sulfuric acid in 1 L water. The resulting mixture was stirred until all solids were dissolved. The aqueous layer was separated and extracted with 2 × 500 mL of diethyl ether. The combined organic extracts were washed sequentially with water and brine, dried over potassium carbonate (500 g) for 30 minutes, filtered, and the filtrate was concentrated in vacuo to give an oil. Sodium bicarbonate (200 mg) was added, the crude material was distilled and the fractions boiling near 100 ° C. were collected to give the title compound (23 grams) with methyl ethyl ketone and 2-butanol as minor impurities. ¹ H NMR (500 MHz, deuterated chloroform) δ 0.45 (app. T, J = 6.59 Hz, 2 H), 0.77
(app. t, J = 5.61 Hz, 2 H), 1.46 (s, 3 H). The preparation of the title compound is also described in WO09105717.

B) 4-Nitrophenyl carbonate 1-methylcyclopropyl 1-methylcyclopropanol (10 g, 137 mmol), 4-nitrophenyl chloroformate (32 g, 152 mmol), and a few pieces of crystals of 4-dimethylaminopyridine (150 mg, 1 .2 mmol) in dichloromethane (462 mL) was cooled to 0 ° C. Triethylamine (36.5 g, 361 mmol) was added dropwise. After 10 minutes, the ice bath was removed and the reaction was stirred at room temperature for 14 hours. The reaction mixture was washed twice with a saturated aqueous sodium carbonate solution. The aqueous phase was extracted with dichloromethane. The combined organic extracts were washed with water, dried over magnesium sulfate, filtered and the filtrate was concentrated in vacuo. The residue is purified by flash silica gel chromatography eluting with a gradient mixture consisting of ethyl acetate heptane solution (constant composition of 0-5% ethyl acetate then 5% ethyl acetate to heptane in the first 10 minutes). To give 20.8 g of the desired carbonate as a clear oil. The oil solidified on standing.
¹ H NMR
(500 MHz, deuterated chloroform) δ 0.77
(app. t, J = 6.59 Hz, 2 H), 1.09 (app. t, J = 7.07 Hz, 2 H), 1.67 (s, 3 H), 7.40
(app.dt, J = 9.27, 3.17 Hz, 2 H), 8.29 (app.dt, J = 9.27, 3.17 Hz, 2 H).

Alternatively, 1-methylcyclopropanol can also be prepared as follows.
1-methylcyclopropanol A 2000 mL four-necked flask was equipped with a mechanical stirrer, an inert gas inlet, a thermometer, and two pressure equalizing dropping funnels. The flask was flushed with nitrogen and charged with 490 mL of diethyl ether followed by 18.2 mL (30 mmol) of titanium tetra (2-ethylhexyl oxide). One dropping funnel was charged with a solution prepared by diluting 28.6 mL (360 mmol) of methyl acetate to 120 mL with ether. The second dropping funnel was charged with 200 mL of 3M ethylmagnesium bromide ether solution. The reaction flask was cooled with an ice-water bath to keep the internal temperature below 10 ° C. 40 ml of methyl acetate solution was added to the flask. The Grignard reagent was then added dropwise from the addition funnel at about 2 drops per second and at a rate of 2 mL or less per minute. After the first 40 mL of Grignard reagent was added, another 20 mL portion of acetic acid methyl ether solution was added. After the second 40 mL of Grignard reagent was added, another 20 mL portion of methyl acetate in diethyl ether was added. A third 40 mL Grignard reagent was added followed by another 20 mL portion of acetic acid methyl ether solution. A fourth 40 mL of Grignard reagent was added followed by the final 20 mL portion of acetic acid methyl ether solution. Following completion of the Grignard reagent addition, the mixture was stirred for an additional 15 minutes. The mixture was then poured into a mixture of 660 g ice and 60 mL concentrated sulfuric acid with rapid stirring to dissolve all solids. The phases were separated and the aqueous phase was extracted again with 50 mL diethyl ether. The combined ether extracts were washed with 15 mL of 10% aqueous sodium carbonate, 15 mL brine, and dried with 30 grams of magnesium sulfate for 1 hour with stirring. The ether solution was then filtered. Tri-n-butylamine (14.3 mL, 60 mmol) and mesitylene (10 mL) were added. Most of the diethyl ether was removed by distillation at atmospheric pressure using a 2.5 cm x 30 cm jacketed Vigreux column. The remaining liquid was transferred to a smaller distillation flask using 2 portions of 10 mL hexane for smooth transfer. Distillation at atmospheric pressure was continued using a 2 cm × 20 cm jacketed Vigreux column. The liquid distilled at 98-105 ° C. was collected to give 14 g of the title compound as a colorless liquid.
¹ H NMR
(400 MHz, deuterated chloroform) δ 0.42-
0.48 (m, 2 H), 0.74-0.80 (m, 2 H), 1.45 (s, 3 H), 1.86 (br. S., 1 H).

Preparation 8: tert-butyl 4-[(6-chloro-5-methylpyrimidin-4-yl) oxy] piperidine-1-carboxylate

２０ｍＬのＢｉｏｔａｇｅ（商標）マイクロ波対応管を窒素パージし、４，６−ジクロロ−５−メチルピリミジン（０．６００ｇ、２．９８ｍｍｏｌ）および４−ヒドロキシピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（５３４ｍｇ、３．２８ｍｍｏｌ）を投入した。１，４−ジオキサン（１４．９ｍＬ）を加え、混合物を１００℃に加熱した。混合物にナトリウムビス（トリメチルシリル）アミド（３．５８ｍＬ、３．５８ｍｍｏｌ、１．０Ｍテトラヒドロフラン溶液）を１０分かけて滴下添加した。混合物を６０分間撹拌し、次いで室温で１２時間撹拌した。水で反応を失活させ、水層を酢酸エチルで３回抽出した。有機抽出物を合わせて硫酸ナトリウムで乾燥させ、濾過し、濾液を真空中で濃縮した。未精製材料をシリカゲルクロマトグラフィー（４０ｇのＳｉＯ_２カラム、０〜５０％の酢酸エチルヘプタン溶液の勾配）によって精製して、表題化合物（８４２ｍｇ、８６％）を得た。

A 20 mL Biotage ™ microwave counterpart tube was purged with nitrogen, and 4,6-dichloro-5-methylpyrimidine (0.600 g, 2.98 mmol) and tert-butyl 4-hydroxypiperidine-1-carboxylate (534 mg, 3.28 mmol) was charged. 1,4-Dioxane (14.9 mL) was added and the mixture was heated to 100 ° C. Sodium bis (trimethylsilyl) amide (3.58 mL, 3.58 mmol, 1.0 M tetrahydrofuran solution) was added dropwise to the mixture over 10 minutes. The mixture was stirred for 60 minutes and then at room temperature for 12 hours. The reaction was quenched with water, and the aqueous layer was extracted 3 times with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo. The crude material was purified by silica gel chromatography (40 g SiO ₂ column, gradient of 0-50% ethyl acetate heptane solution) to give the title compound (842 mg, 86%).

Preparation 9: tert-butyl (3R, 4S) -4-[(6-chloro-5-methylpyrimidin-4-yl) oxy] -3-fluoropiperidine-1-carboxylate (racemic)

（３，４−シス）−３−フルオロ−４−ヒドロキシ−ピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（ラセミ体）（調製例１）（１．０ｇ、４．６ｍｍｏｌ）および４，６−ジクロロ−５−メチルピリミジン（８１８ｍｇ、５．０２ｍｍｏｌ）を無水テトラヒドロフラン（２３ｍＬ）に溶かした溶液に、水素化ナトリウム（２０１ｍｇ、５．０２ｍｍｏｌ、６０％鉱油分散液）を２回に分けて０℃で加えた。１８時間後、反応混合物を飽和塩化アンモニウム水溶液で失活させ、水で希釈した。得られる混合物を酢酸エチルで３回抽出した。有機層を合わせて硫酸ナトリウムで乾燥させ、濾過し、濾液を減圧下で濃縮して、表題化合物を淡黄色の油状物（１．５６ｇ、９９％）として得た。
¹H NMR
(400 MHz, 重水素化クロロホルム) δ 1.46
(s, 9 H), 1.84 - 1.91 (m, 1 H), 2.04 - 2.17 (m, 1 H), 2.24 (s, 3 H), 3.09 -
3.22 (m, 1 H), 3.29 - 3.43 (m, 1 H), 3.78 - 4.01 (m, 1 H), 4.09 - 4.20 (m, 1
H), 4.74 - 4.93 (m, 1 H), 5.31 - 5.43 (m, 1 H), 8.36 (s, 1 H).LCMS (ES+): 346.4
(M+1).

Tert-Butyl (racemic) (3,4-cis) -3-fluoro-4-hydroxy-piperidine-1-carboxylate (Preparation Example 1) (1.0 g, 4.6 mmol) and 4,6-dichloro- Sodium hydride (201 mg, 5.02 mmol, 60% mineral oil dispersion) was added in two portions at 0 ° C. to a solution of 5-methylpyrimidine (818 mg, 5.02 mmol) in anhydrous tetrahydrofuran (23 mL). . After 18 hours, the reaction mixture was quenched with saturated aqueous ammonium chloride and diluted with water. The resulting mixture was extracted with ethyl acetate three times. The combined organic layers were dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the title compound as a pale yellow oil (1.56 g, 99%).
¹ H NMR
(400 MHz, deuterated chloroform) δ 1.46
(s, 9 H), 1.84-1.91 (m, 1 H), 2.04-2.17 (m, 1 H), 2.24 (s, 3 H), 3.09-
3.22 (m, 1 H), 3.29-3.43 (m, 1 H), 3.78-4.01 (m, 1 H), 4.09-4.20 (m, 1
H), 4.74-4.93 (m, 1 H), 5.31-5.43 (m, 1 H), 8.36 (s, 1 H) .LCMS (ES +): 346.4
(M + 1).

Preparation Example 10: 4-Chloro-6-{[(3R, 4S) -3-fluoropiperidin-4-yl] oxy} -5-methylpyrimidine

（３Ｒ，４Ｓ）−４−［（６−クロロ−５−メチルピリミジン−４−イル）オキシ］−３−フルオロピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（１．４ｇ、４．０ｍｍｏｌ）の無水１，２−ジクロロエタン（２０ｍＬ）溶液に、陽圧窒素流のもとで、室温でトリフルオロ酢酸（４．０ｍＬ、５２．０ｍｍｏｌ）を加えた。２時間後、減圧下で揮発性物質を除去し、加熱して無色の残渣を得た。残渣をジクロロメタンに溶かし、飽和炭酸水素ナトリウム水溶液で塩基性化した。次いで混合物をジクロロメタンで３回抽出した。有機層を合わせて硫酸ナトリウムで乾燥させ、濾過し、減圧下で濃縮して、生成物をオフホワイトの固体（９３０ｍｇ、９３％）として得た。1H NMR (400 MHz, 重水素化クロロホルム) δ 1.88 - 2.07 (m, 2 H) 2.25 (s, 3 H) 2.73 - 2.82 (m, 1 H) 2.86 - 2.99
(m, 1 H) 3.12 - 3.20 (m, 1 H) 3.31 - 3.39 (m, 1 H) 4.76 - 4.93 (m, 1 H) 5.24 -
5.37 (m, 1 H) 8.36 (s, 1 H) LCMS: (ES+): 246.2 (M+1).

Anhydrous 1 of (3R, 4S) -4-[(6-Chloro-5-methylpyrimidin-4-yl) oxy] -3-fluoropiperidine-1-carboxylate (1.4 g, 4.0 mmol) , 2-Dichloroethane (20 mL) was added trifluoroacetic acid (4.0 mL, 52.0 mmol) at room temperature under a stream of positive pressure nitrogen. After 2 hours, volatiles were removed under reduced pressure and heated to give a colorless residue. The residue was dissolved in dichloromethane and basified with saturated aqueous sodium bicarbonate. The mixture was then extracted 3 times with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give the product as an off-white solid (930 mg, 93%). 1H NMR (400 MHz, deuterated chloroform) δ 1.88-2.07 (m, 2 H) 2.25 (s, 3 H) 2.73-2.82 (m, 1 H) 2.86-2.99
(m, 1 H) 3.12-3.20 (m, 1 H) 3.31-3.39 (m, 1 H) 4.76-4.93 (m, 1 H) 5.24-
5.37 (m, 1 H) 8.36 (s, 1 H) LCMS: (ES +): 246.2 (M + 1).

Preparation 11: (3R, 4S) -4-[(6-Chloro-5-methylpyrimidin-4-yl) oxy] -3-fluoropiperidine-1-carboxylic acid 1-methylcyclopropyl

４−クロロ−６−｛［（３Ｒ，４Ｓ）−３−フルオロピペリジン−４−イル］オキシ｝−５−メチルピリミジン（９２５ｍｇ、３．７６ｍｍｏｌ）およびトリエチルアミン（１．５７ｍＬ、１１．３ｍｍｏｌ）をジクロロメタン（２０．０ｍＬ）に溶かした溶液に、４−ニトロフェニル炭酸１−メチルシクロプロピル（１．７９ｍｇ、７．５３ｍｍｏｌ）を室温で加えた。７２時間後、水で反応を失活させ、ジクロロメタンで３回抽出した。有機層を合わせて飽和炭酸水素ナトリウム水溶液で黄色が消えるまで継続的に洗浄した。次いで有機層を硫酸ナトリウムで乾燥させ、濾過し、減圧下で濃縮した。得られる未精製残渣をフラッシュクロマトグラフィー（シリカ：１０〜５０％の酢酸エチル：ヘプタン）によって精製して、８３０ｍｇ（６４％）の所望の生成物を白色の固体として得た。1H NMR (500 MHz, 重水素化クロロホルム) δ 0.63 - 0.68 (m, 2 H), 0.87 - 0.94 (m, 2 H), 1.60 (s, 3 H), 1.86 -
1.97 (m, 1 H), 2.08 - 2.19 (m, 1 H), 2.27 (s, 3 H), 3.11 - 3.27 (m, 1 H) 3.27 -
3.49 (m, 1 H), 3.78 - 4.11 (m, 1 H), 4.11 - 4.27 (m, 1 H), 4.77 - 4.96 (m, 1
H), 5.33 - 5.46 (m, 1 H), 8.40 (s, 1 H) LCMS: (ES+): 344.4 (M+1).

4-Chloro-6-{[(3R, 4S) -3-fluoropiperidin-4-yl] oxy} -5-methylpyrimidine (925 mg, 3.76 mmol) and triethylamine (1.57 mL, 11.3 mmol) in dichloromethane To a solution dissolved in (20.0 mL), 4-nitrophenyl carbonate 1-methylcyclopropyl carbonate (1.79 mg, 7.53 mmol) was added at room temperature. After 72 hours, the reaction was quenched with water and extracted three times with dichloromethane. The organic layers were combined and washed with saturated aqueous sodium bicarbonate until the yellow color disappeared. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting crude residue was purified by flash chromatography (silica: 10-50% ethyl acetate: heptane) to give 830 mg (64%) of the desired product as a white solid. 1H NMR (500 MHz, deuterated chloroform) δ 0.63-0.68 (m, 2 H), 0.87-0.94 (m, 2 H), 1.60 (s, 3 H), 1.86-
1.97 (m, 1 H), 2.08-2.19 (m, 1 H), 2.27 (s, 3 H), 3.11-3.27 (m, 1 H) 3.27-
3.49 (m, 1 H), 3.78-4.11 (m, 1 H), 4.11-4.27 (m, 1 H), 4.77-4.96 (m, 1
H), 5.33-5.46 (m, 1 H), 8.40 (s, 1 H) LCMS: (ES +): 344.4 (M + 1).

Preparation Example 12: 2,3-Dihydro-1H-imidazo [1,2-b] pyrazole

５−アミノピラゾール（３６ｇ、０．４３ｍｏｌ）の１，４−ジオキサン（１Ｌ）溶液に、１，２−ジブロモエタン（１０６ｇ、０．５６ｍｏｌ）を加えた。反応混合物に、撹拌しながら周囲温度でトリエチルアミン（１４６ｍＬ、１．０４ｍｏｌ）を加えた。得られる混合物を１８時間還流加熱した。反応混合物を室温に冷却し、沈殿を濾過によって除去した。濾液を減圧下で濃縮した。粗生成物を、５％のメタノールジクロロメタン溶液からなる定組成混合物を使用するシリカゲルカラムクロマトグラフィーによって精製して、表題化合物を白色の固体（９．６グラム、２０％）として得た。
¹H NMR
(500 MHz, 重水素化ジメチルスルホキシド) δ
7.15 (1 H, d, J=1.71 Hz), 5.59 (1 H, br. s.), 5.20 (1 H, d, J=1.71 Hz), 3.96 -
4.05 (2 H, m), 3.78 - 3.85 (2 H, m)

1,2-Dibromoethane (106 g, 0.56 mol) was added to a solution of 5-aminopyrazole (36 g, 0.43 mol) in 1,4-dioxane (1 L). To the reaction mixture was added triethylamine (146 mL, 1.04 mol) at ambient temperature with stirring. The resulting mixture was heated to reflux for 18 hours. The reaction mixture was cooled to room temperature and the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using an isocratic mixture consisting of 5% methanol in dichloromethane to give the title compound as a white solid (9.6 grams, 20%).
¹ H NMR
(500 MHz, deuterated dimethyl sulfoxide) δ
7.15 (1 H, d, J = 1.71 Hz), 5.59 (1 H, br. S.), 5.20 (1 H, d, J = 1.71 Hz), 3.96-
4.05 (2 H, m), 3.78-3.85 (2 H, m)

  The synthesis of the title compound is also described in US 2998537.

Preparation Example 13: 4- (6- (2,3-Dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yloxy) piperidine-1-carboxylic acid tert- Butyl

Ｂｉｏｔａｇｅ（商標）マイクロ波対応バイアルにおいて、４−［（６−クロロ−５−メチルピリミジン−４−イル）オキシ］ピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（４９ｍｇ、０．１５ｍｍｏｌ）（調製例８）および２，３−ジヒドロ−１Ｈ−イミダゾ［１，２−ｂ］ピラゾール（調製例１２）（１７ｍｇ、０．１６ｍｍｏｌ）を１ｍＬのＮ−メチルピロリドンに溶解させた。混合物に炭酸セシウム（２００ｍｇ、０．６１ｍｍｏｌ）を加え、バイアルを窒素パージした。次いで反応混合物を１５０℃で５時間撹拌した。水で反応を失活させ、水層を酢酸エチルで３回抽出した。有機層を合わせて硫酸ナトリウムで乾燥させ、濾過し、真空中で濃縮した。未精製材料を、シリカゲルクロマトグラフィー（０％〜１００％の酢酸エチルヘプタン溶液の勾配）によって精製して、残存するＮ−メチルピロリドンが混入した所望の生成物を得た（６０ｍｇ）。LCMS (ES+): 401.5 (M+H).

Biotage ™ microwave compatible vial in tert-butyl 4-[(6-chloro-5-methylpyrimidin-4-yl) oxy] piperidine-1-carboxylate (49 mg, 0.15 mmol) (Preparation 8) And 2,3-dihydro-1H-imidazo [1,2-b] pyrazole (Preparation Example 12) (17 mg, 0.16 mmol) were dissolved in 1 mL of N-methylpyrrolidone. To the mixture was added cesium carbonate (200 mg, 0.61 mmol) and the vial was purged with nitrogen. The reaction mixture was then stirred at 150 ° C. for 5 hours. The reaction was quenched with water, and the aqueous layer was extracted 3 times with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by silica gel chromatography (gradient from 0% to 100% ethyl acetate heptane solution) to give the desired product contaminated with residual N-methylpyrrolidone (60 mg). LCMS (ES +): 401.5 (M + H).

Preparation Example 14: 1- (6-Chloro-5-methylpyrimidin-4-yl) -2,3-dihydro-1H-imidazo [1,2-b] pyrazole

２，３−ジヒドロ−１Ｈ−イミダゾ［１，２−ｂ］ピラゾール（調製例１２）（１４．１ｇ、１２９ｍｍｏｌ）および４，６−ジクロロ−５−メチルピリミジン（２１．１ｇ、１２９ｍｍｏｌ）を８００ｍＬのテトラヒドロフランに溶かした、０℃に冷却した溶液に、ナトリウムビス（トリメチルシリル）アミド（１Ｍテトラヒドロフラン溶液、１３８ｍＬ、１３８ｍｍｏｌ）を１時間かけて滴下添加した。２時間後、水を加え、テトラヒドロフランを蒸発させた。次いでジクロロメタンを加え、水相をジクロロメタンで３回抽出した。有機層を合わせて硫酸マグネシウムで乾燥させ、濾過し、濾液を真空中で濃縮した。残渣を最小量のジクロロメタンに溶解させ、ヘプタンを使用して所望の生成物を沈殿させた。得られる固体を濾過し、ヘプタンで洗浄し、真空中で乾燥させて、２４．６ｇ（収率９０％）のベージュ色の固体を得た。LCMS (ES+): 236.3 (M+1).

2,3-Dihydro-1H-imidazo [1,2-b] pyrazole (Preparation Example 12) (14.1 g, 129 mmol) and 4,6-dichloro-5-methylpyrimidine (21.1 g, 129 mmol) in 800 mL Sodium bis (trimethylsilyl) amide (1M tetrahydrofuran solution, 138 mL, 138 mmol) was added dropwise to the solution dissolved in tetrahydrofuran and cooled to 0 ° C. over 1 hour. After 2 hours, water was added and the tetrahydrofuran was evaporated. Dichloromethane was then added and the aqueous phase was extracted 3 times with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and the filtrate was concentrated in vacuo. The residue was dissolved in a minimum amount of dichloromethane and the desired product was precipitated using heptane. The resulting solid was filtered, washed with heptane and dried in vacuo to give 24.6 g (90% yield) of a beige solid. LCMS (ES +): 236.3 (M + 1).

Preparation 15: (3S, 4R) -4- (6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yloxy) -3 -Tert-butyl fluoropiperidine-1-carboxylate

Ｂｉｏｔａｇｅ（商標）マイクロ波対応バイアルにおいて、１−（６−クロロ−５−メチルピリミジン−４−イル）−２，３−ジヒドロ−１Ｈ−イミダゾ［１，２−ｂ］ピラゾール（調製例１４）（１０５ｍｇ、０．４４６ｍｍｏｌ）および（３Ｓ，４Ｒ）−３−フルオロ−４−ヒドロキシシクロヘキサンカルボン酸ｔｅｒｔ−ブチル（調製例１）（１１２ｍｇ、０．５１１ｍｍｏｌ）をテトラヒドロフラン（４．５ｍＬ）に溶解させた。溶液を６０℃まで加熱し、カリウムｔｅｒｔ−ブトキシドを加えた（０．７１ｍＬ、０．７１ｍｍｏｌ）。４日後、水を加え、反応混合物をジクロロメタンで希釈した。水層をジクロロメタンで３回抽出し、有機層を合わせて硫酸マグネシウムで乾燥させ、濾過し、濾液を真空中で濃縮した。未精製材料をシリカゲルでのカラムクロマトグラフィー（４０％〜１００％の酢酸エチル／ヘプタン）によって精製して、１００ｍｇ（収率５４％）の表題化合物を白色の固体として得た。LCMS (ES+): 419.6 (M+1).

In a Biotage ™ microwave ready vial, 1- (6-chloro-5-methylpyrimidin-4-yl) -2,3-dihydro-1H-imidazo [1,2-b] pyrazole (Preparation Example 14) ( 105 mg, 0.446 mmol) and tert-butyl (3S, 4R) -3-fluoro-4-hydroxycyclohexanecarboxylate (Preparation Example 1) (112 mg, 0.511 mmol) were dissolved in tetrahydrofuran (4.5 mL). The solution was heated to 60 ° C. and potassium tert-butoxide was added (0.71 mL, 0.71 mmol). After 4 days, water was added and the reaction mixture was diluted with dichloromethane. The aqueous layer was extracted 3 times with dichloromethane, the combined organic layers were dried over magnesium sulfate, filtered and the filtrate was concentrated in vacuo. The crude material was purified by column chromatography on silica gel (40% -100% ethyl acetate / heptane) to give 100 mg (54% yield) of the title compound as a white solid. LCMS (ES +): 419.6 (M + 1).

Example 1
4-{[6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} piperidine-1-carboxylate

４−［（６−クロロ−５−メチルピリミジン−４−イル）オキシ］ピペリジン−１−カルボン酸イソプロピル（調製例６）（０．０２０ｇ、０．０５６ｍｍｏｌ）のＮ−メチルピロリジノン（０．５６ｍＬ）溶液に、２，３−ジヒドロ−１Ｈ−イミダゾ［１，２−ｂ］ピラゾール（調製例１２）（６ｍｇ、０．０６ｍｍｏｌ）に続いて炭酸セシウム（９１ｍｇ、０．２８ｍｍｏｌ）を加えた。混合物を３時間１５０℃に加熱した。反応液を水で希釈し、水層をジクロロメタンで３回抽出し、有機層を合わせて硫酸ナトリウムで乾燥させ、濾過し、真空中で濃縮した。未精製材料の精製を、７．０分かけて８０％の水／２０％のアセトニトリルから４０％の水／６０％のアセトニトリルへの直線勾配で溶離し、次いで７．０〜７．５分で０％の水／１００％のアセトニトリルへと勾配をかけ、８．５分まで０％の水／１００％のアセトニトリルで保持する（０．０３％の水酸化アンモニウム改質剤）Ｗａｔｅｒｓ ＸＢｒｉｄｇｅ Ｃ_１８ １９×１００ｍｍ、５マイクロメートルカラムでの逆相ＨＰＬＣ、流量２５ｍＬ／分によって行って、表題化合物（９．１ｍｇ、４２％）を得た。分析ＬＣＭＳ：保持時間１．０４分（Ｗａｔｅｒｓ Ａｃｑｕｉｔｙ ＨＳＳ Ｔ３ ２．１×５０ｍｍ １．８ｕｍカラム、１．６分かけて９５％の水／５％のアセトニトリルから２％の水／９８％のアセトニトリルへの直線勾配、次いで１．８分まで２％の水／９８％のアセトニトリルで保持、０．０５％のトリフルオロ酢酸改質剤、流量１．３ｍＬ／分）、LCMS (ES+): 387.5 (M+H).

4-[(6-Chloro-5-methylpyrimidin-4-yl) oxy] piperidine-1-carboxylate (Preparation Example 6) (0.020 g, 0.056 mmol) in N-methylpyrrolidinone (0.56 mL) To the solution was added 2,3-dihydro-1H-imidazo [1,2-b] pyrazole (Preparation Example 12) (6 mg, 0.06 mmol) followed by cesium carbonate (91 mg, 0.28 mmol). The mixture was heated to 150 ° C. for 3 hours. The reaction was diluted with water and the aqueous layer was extracted three times with dichloromethane, the combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Purification of the crude material was eluted with a linear gradient from 80% water / 20% acetonitrile to 40% water / 60% acetonitrile over 7.0 minutes, then at 7.0-7.5 minutes. Gradient to 0% water / 100% acetonitrile and hold with 0% water / 100% acetonitrile for up to 8.5 minutes (0.03% ammonium hydroxide modifier) Waters XBridge C ₁₈ 19 Performed by reverse phase HPLC on x100 mm, 5 micrometer column, flow rate 25 mL / min to give the title compound (9.1 mg, 42%). Analytical LCMS: Retention time 1.04 min (Waters Acquity HSS T3 2.1 × 50 mm 1.8 um column, 95% water / 5% acetonitrile to 2% water / 98% acetonitrile over 1.6 min. Linear gradient, then hold with 2% water / 98% acetonitrile until 1.8 min, 0.05% trifluoroacetic acid modifier, flow rate 1.3 mL / min, LCMS (ES +): 387.5 (M + H).

(Example 2)
4-{[6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} piperidine-1-carboxylate 1-methyl Cyclopropyl

４−（６−（２，３−ジヒドロ−１Ｈ−イミダゾ［１，２−ｂ］ピラゾール−１−イル）−５−メチルピリミジン−４−イルオキシ）ピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（調製例１３）（６０ｍｇ、０．１５ｍｍｏｌ）を３ｍＬのジクロロメタンに溶かした溶液に、０．４ｍＬの塩酸（４Ｍ １，４−ジオキサン溶液）を加えた。混合物を室温で１２時間撹拌した。減圧下で溶媒を蒸発させ、残渣を高真空中で乾燥させた。次いで残渣をジクロロメタン（３ｍＬ）に溶解させ、トリエチルアミン（０．１２５ｍＬ、０．９０ｍｍｏｌ）を加えた後、４−ニトロフェニル炭酸１−メチルシクロプロピル（７１．２ｍｇ、０．３ｍｍｏｌ）を加えた。フラスコを窒素パージし、次いで反応混合物を室温で４８時間撹拌した。反応液をジクロロメタンで希釈し、水で失活させた。水相をジクロロメタンで２回抽出し、有機層を合わせて飽和炭酸水素ナトリウム水溶液、続いてブラインで洗浄した。有機相を硫酸マグネシウムで乾燥させ、濾過し、濾液を真空中で濃縮し、高真空中で乾燥させて、１４８ｍｇの未精製材料を得た。この材料の一部（４９ｍｇ）をジメチルスルホキシド（０．９ｍＬ）に溶解させ、水アセトニトリル溶液の勾配（０．０３％の水酸化アンモニウム改質剤）で溶離を行うＷａｔｅｒｓ ＸＢｒｉｄｇｅ Ｃ_１８ カラム１９×１００ｍｍ、５マイクロメートルカラムでの分取ＨＰＬＣによって精製した。分析ＬＣＭＳ：保持時間３．０９分（Ｗａｔｅｒ Ａｔｌａｎｔｉｓ Ｃ_１８４．６×５０ｍｍ、５μｍカラム、４分かけて９５％の水／アセトニトリルから５％の水／アセトニトリルへの直線勾配、０．０５％のトリフルオロ酢酸改質剤、流量２．０ｍＬ／分）、LCMS (ES+): 399.2 (M+H).

4- (6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yloxy) piperidine-1-carboxylate tert-butyl (Preparation Example 13) To a solution of (60 mg, 0.15 mmol) in 3 mL of dichloromethane was added 0.4 mL of hydrochloric acid (4M 1,4-dioxane solution). The mixture was stirred at room temperature for 12 hours. The solvent was evaporated under reduced pressure and the residue was dried in high vacuum. The residue was then dissolved in dichloromethane (3 mL) and triethylamine (0.125 mL, 0.90 mmol) was added followed by 4-nitrophenyl carbonate 1-methylcyclopropyl (71.2 mg, 0.3 mmol). The flask was purged with nitrogen and then the reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was diluted with dichloromethane and quenched with water. The aqueous phase was extracted twice with dichloromethane and the combined organic layers were washed with saturated aqueous sodium bicarbonate followed by brine. The organic phase was dried over magnesium sulfate, filtered and the filtrate was concentrated in vacuo and dried in high vacuum to give 148 mg of crude material. A portion of this material (49 mg) is dissolved in dimethyl sulfoxide (0.9 mL) and a Waters XBridge C ₁₈ column 19 × 100 mm eluting with a gradient of water acetonitrile solution (0.03% ammonium hydroxide modifier). Purified by preparative HPLC on a 5 micrometer column. Analytical LCMS: retention time 3.09 min (Water Atlantis C ₁₈ 4.6 × 50 mm, 5 μm column, linear gradient from 95% water / acetonitrile to 5% water / acetonitrile over 4 min, 0.05% Trifluoroacetic acid modifier, flow rate 2.0 mL / min), LCMS (ES +): 399.2 (M + H).

(Example 3)
(3R, 4S) -4-{[6- (2,3-Dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} -3- Fluoropiperidine-1-carboxylic acid 1-methylcyclopropyl (racemic)

Ｂｉｏｔａｇｅ（商標）マイクロ波対応バイアルにおいて、（３Ｒ，４Ｓ）−４−（６−クロロ−５−メチルピリミジン−４−イルオキシ）−３−フルオロピペリジン−１−カルボン酸１−メチルシクロプロピル（ラセミ体）（調製例１１）（２８ｍｇ、０．０８１ｍｍｏｌ）および２，３−ジヒドロ−１Ｈ−イミダゾ［１，２−ｂ］ピラゾール（調製例１２）（１０．６ｍｇ、０．０９７ｍｍｏｌ）を１ｍＬのＮ−メチルピロリドンに溶解させた。この混合物に炭酸セシウム（１３２ｍｇ、０．４０５ｍｍｏｌ）を加え、バイアルを窒素パージした。次いで反応混合物を１５０℃で３時間撹拌した。反応液を水で失活させ、水層を酢酸エチルで３回抽出した。有機層を合わせて硫酸ナトリウムで乾燥させ、濾過し、濾液を真空中で濃縮して、５０ｍｇの未精製物を得た。この材料をジメチルスルホキシド（０．９ｍＬ）に溶解させ、水アセトニトリル溶液の勾配（０．０３％の水酸化アンモニウム改質剤）で溶離を行うＷａｔｅｒｓ ＸＢｒｉｄｇｅ Ｃ_１８カラム１９×１００ｍｍ、５マイクロメートルカラムでの分取ＨＰＬＣによって精製した。分析ＬＣＭＳ：保持時間２．９７分（Ｗａｔｅｒ Ａｔｌａｎｔｉｓ Ｃ_１８ ４．６×５０ｍｍ、５μｍカラム、４分かけて９５％の水／アセトニトリルから５％の水／アセトニトリルへの直線勾配、０．０５％のトリフルオロ酢酸改質剤、流量２．０ｍＬ／分）、LCMS (ES+): 417.1 (M+H).

In a Biotage ™ microwave compatible vial, (3R, 4S) -4- (6-chloro-5-methylpyrimidin-4-yloxy) -3-fluoropiperidine-1-carboxylate 1-methylcyclopropyl (racemic) ) (Preparation Example 11) (28 mg, 0.081 mmol) and 2,3-dihydro-1H-imidazo [1,2-b] pyrazole (Preparation Example 12) (10.6 mg, 0.097 mmol) in 1 mL N- Dissolved in methylpyrrolidone. To this mixture was added cesium carbonate (132 mg, 0.405 mmol) and the vial was purged with nitrogen. The reaction mixture was then stirred at 150 ° C. for 3 hours. The reaction solution was quenched with water, and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo to give 50 mg of crude. This material is dissolved in dimethyl sulfoxide (0.9 mL) and eluted with a gradient of water acetonitrile solution (0.03% ammonium hydroxide modifier) on a Waters XBridge C ₁₈ column 19 × 100 mm, 5 micrometer column. Was purified by preparative HPLC. Analytical LCMS: Retention time 2.97 min (Water Atlantis C ₁₈ 4.6 × 50 mm, 5 μm column, linear gradient from 95% water / acetonitrile to 5% water / acetonitrile over 4 min, 0.05% Trifluoroacetic acid modifier, flow rate 2.0 mL / min), LCMS (ES +): 417.1 (M + H).

Example 4
(3R, 4S) -4-{[6- (2,3-Dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} -3- Isopropyl fluoropiperidine-1-carboxylate (racemic)

Ｂｉｏｔａｇｅ（商標）マイクロ波対応バイアルにおいて、（３Ｒ，４Ｓ）−４−（６−クロロ−５−メチルピリミジン−４−イルオキシ）−３−フルオロピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（ラセミ体）（調製例９）（４６ｍｇ、０．１３ｍｍｏｌ）および２，３−ジヒドロ−１Ｈ−イミダゾ［１，２−ｂ］ピラゾール（調製例１２）（１７．５ｍｇ、０．１６ｍｍｏｌ）を１ｍＬのＮ−メチルピロリドンに溶解させた。混合物に炭酸セシウム（２１７ｍｇ、０．６７ｍｍｏｌ）を加え、バイアルを窒素パージした。次いで反応混合物を１５０℃で２２時間撹拌した。水で反応を失活させ、水層を酢酸エチルで３回抽出した。有機層を合わせて硫酸ナトリウムで乾燥させ、濾過し、真空中で濃縮して、未精製の（３Ｒ，４Ｓ）−４−（６−（２，３−ジヒドロ−１Ｈ−イミダゾ［１，２−ｂ］ピラゾール−１−イル）−５−メチルピリミジン−４−イルオキシ）−３−フルオロピペリジン−１−カルボン酸ｔｅｒｔ−ブチル（ラセミ体）を得、これを精製せずに次のステップで使用した。この残渣を３ｍＬのジクロロメタンに溶かした溶液に、０．３３ｍＬの塩酸（４Ｍジオキサン溶液）を加えた。混合物を室温で１２時間撹拌し、トリフルオロ酢酸（０．１６ｍＬ、２．１ｍｍｏｌ）を加え、次いで３時間撹拌した。溶媒を蒸発させ、トルエン共沸混合物によってトリフルオロ酢酸を除去した。次いで残渣をジクロロメタン（２ｍＬ）に溶解させ、Ｎ−Ｎ−ジイソプロピルエチルアミン（０．２ｍＬ、１ｍｍｏｌ）を加えた。溶液を０℃に冷却し、１Ｍのクロロギ酸イソプロピルトルエン溶液（０．６８ｍＬ、０．７ｍｍｏｌ）を加えた。反応混合物を窒素中にて室温で１２時間撹拌した。反応液をジクロロメタンで希釈し、水で失活させた。水相をジクロロメタンで抽出し、有機層を合わせて硫酸マグネシウムで乾燥させ、濾過し、蒸発にかけ、高真空中で乾燥させて、５０ｍｇの未精製物を得た。この材料をジメチルスルホキシド（０．９ｍＬ）に溶解させ、水アセトニトリル溶液の勾配（０．０３％の水酸化アンモニウム改質剤）で溶離を行うＷａｔｅｒｓ ＸＢｒｉｄｇｅ Ｃ_１８カラム１９×１００ｍｍ、５μｍカラムでの分取ＨＰＬＣによって精製した。分析ＬＣＭＳ：保持時間２．９５分（Ｗａｔｅｒ Ａｔｌａｎｔｉｓ Ｃ_１８４．６×５０ｍｍ、５μｍカラム、４分かけて９５％の水／アセトニトリルから５％の水／アセトニトリルへの直線勾配、０．０５％のトリフルオロ酢酸改質剤、流量２．０ｍＬ／分）、LCMS (ES+): 405.1 (M+H).

In a Biotage ™ microwave ready vial, tert-butyl (3R, 4S) -4- (6-chloro-5-methylpyrimidin-4-yloxy) -3-fluoropiperidine-1-carboxylate (racemic) ( Preparation 9) (46 mg, 0.13 mmol) and 2,3-dihydro-1H-imidazo [1,2-b] pyrazole (Preparation 12) (17.5 mg, 0.16 mmol) in 1 mL of N-methylpyrrolidone Dissolved in. To the mixture was added cesium carbonate (217 mg, 0.67 mmol) and the vial was purged with nitrogen. The reaction mixture was then stirred at 150 ° C. for 22 hours. The reaction was quenched with water, and the aqueous layer was extracted 3 times with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to give crude (3R, 4S) -4- (6- (2,3-dihydro-1H-imidazo [1,2- b] pyrazol-1-yl) -5-methylpyrimidin-4-yloxy) -3-fluoropiperidine-1-carboxylate tert-butyl (racemate) was obtained and used in the next step without purification. . To a solution of the residue in 3 mL of dichloromethane, 0.33 mL of hydrochloric acid (4M dioxane solution) was added. The mixture was stirred at room temperature for 12 hours, trifluoroacetic acid (0.16 mL, 2.1 mmol) was added and then stirred for 3 hours. The solvent was evaporated and trifluoroacetic acid was removed by toluene azeotrope. The residue was then dissolved in dichloromethane (2 mL) and NN-diisopropylethylamine (0.2 mL, 1 mmol) was added. The solution was cooled to 0 ° C. and 1M isopropyltoluene chloroformate solution (0.68 mL, 0.7 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours under nitrogen. The reaction mixture was diluted with dichloromethane and quenched with water. The aqueous phase was extracted with dichloromethane and the combined organic layers were dried over magnesium sulfate, filtered, evaporated and dried in high vacuum to give 50 mg of crude. This material is dissolved in dimethyl sulfoxide (0.9 mL) and eluted with a gradient of water acetonitrile solution (0.03% ammonium hydroxide modifier) on a Waters XBridge C ₁₈ column 19 × 100 mm, 5 μm column. Purified by preparative HPLC. Analytical LCMS: Retention time 2.95 min (Water Atlantis C ₁₈ 4.6 × 50 mm, 5 μm column, linear gradient from 95% water / acetonitrile to 5% water / acetonitrile over 4 min, 0.05% Trifluoroacetic acid modifier, flow rate 2.0 mL / min), LCMS (ES +): 405.1 (M + H).

(Example 5)
(3S, 4R) -4-{[6- (2,3-Dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} -3- 1-methylcyclopropyl fluoropiperidine-1-carboxylate

(Example 6)
(3R, 4S) -4-{[6- (2,3-Dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} -3- 1-methylcyclopropyl fluoropiperidine-1-carboxylate

実施例５および実施例６の表題化合物は、実施例３のキラル分離によって調製した。

The title compounds of Example 5 and Example 6 were prepared by the chiral separation of Example 3.

The racemic mixture (Example 3) was separated by Analytical SFC-2 on a Chiralpak AD-H column 10 × 250 mm, mobile phase 75/25 carbon dioxide / methanol, flow rate 10.0 mL / min. UV detection 210 nm. Peak 1 (Example 5): 217 mg, yield 30%, retention time 5.16 minutes. Peak 2 (Example 6): 206 mg, yield 29%, retention time 6.32 minutes. LCMS (ES +): 417.4 (M + H). ¹ H NMR (500 MHz, deuterated chloroform) δ 0.59-0.72 (m, 2 H), 0.85-0.96 (m, 2
H), 1.58 (s, 3 H), 1.91 (br. S., 1 H), 2.14 (br. S., 1 H), 2.20 (s, 3 H), 3.17
(br. s., 1 H), 3.37 (br. s., 1 H), 4.02-4.28 (m, 2 H), 4.36-4.45 (m, 2 H),
4.59-4.68 (m, 2 H), 4.78-4.98 (m, 1 H), 5.32-5.46 (m, 1 H), 5.73 (s, 1
H), 7.44 (d, J = 1.5 Hz, 1 H), 8.33 (s, 1 H).

  The absolute stereochemistry of Examples 5 and 6 is based on tert-butyl (3S, 4R) -3-fluoro-4-hydroxypiperidine-1-carboxylate (Enantiomer 1, Preparation 1, Step D). , (3S, 4R) -4- (6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yloxy) -3-fluoropiperidine Tert-Butyl-1-carboxylate (Preparation Example 15) was assigned through the steps of Preparation Examples 9-11 to give Example 5.

  Throughout this application, various publications are cited as references. The disclosures of those publications are incorporated herein by reference in their entirety for all purposes.

  It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims (19)

Compound having formula I

[Where:
X is A or B

And
Y is O or a bond;
R ¹ is —C (O) —O—R ³ or

And
R ² is hydrogen, cyano, C ₁ -C ₆ alkyl, or C ₃ -C ₆ cycloalkyl,
R ³ is substituted with C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, or C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ fluoroalkyl, halo, or hydroxy C ₃ -C ₆ cycloalkyl, provided that the halo, C ₁ -C ₆ alkoxy, or hydroxy group is not bonded at the carbon atom connected to O in R ¹ ;
R ⁴ is C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkyl, halo, cyano, or C ₃ -C ₆ cycloalkyl,
R ⁵ is hydrogen, cyano, nitro, C ₁ -C ₆ fluoroalkyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ fluoroalkoxy, or C ₃ -C ₆ cycloalkyl. ,
R ⁶ is _hydrogen, C 1 -C _{6 alkyl,} C 3 -C ₆ cycloalkyl, -C (O) -NH ₂ or _C 1 -C ₆ alkyl substituted with hydroxy or _C 1 -C ₆ alkoxy, And
R ^7a and R ^7b are each independently hydrogen, fluoro, or C ₁ -C ₆ alkyl;
m is 1 or 2, and when m is 1, R ⁸ is hydrogen, C ₁ -C ₆ alkyl, —CH ₂ — (C ₁ -C ₅ ) haloalkyl, C ₃ -C ₆ cycloalkyl, or hydroxy a _C 1 -C ₆ alkyl which is substituted, when m is 2, each ^{R 8} is _{independently,} C 1 -C ₃ alkyl or _{-CH 2 - (C 1 ~C 2} ) Is haloalkyl]
Or a pharmaceutically acceptable salt thereof. The compound of claim ¹ , wherein X is A and R ¹ is —C (O) —O—R ³ . The compound according to claim 1 or 2, wherein R ⁶ and R ⁸ are each hydrogen. R ³ _{is C} 3 -C ₆ cycloalkyl substituted with C 1 -C ₃ alkyl, A compound according to claim 1, 2 or 3. The compound according to any one of claims 1 to 4, wherein R ^7a and R ^7b are each independently hydrogen, fluoro, or C ₁ -C ₃ alkyl. R ² is hydrogen, ^{R 5} is _C 1 -C ₆ alkyl, A compound according to any one of claims 1 to 5. The following compounds:
Isopropyl 4-{[6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} piperidine-1-carboxylate;
1-methylcyclopropyl 4-{[6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} piperidine-1- Carboxylate;
1-methylcyclopropyl (3R, 4S) -4-{[6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] Oxy} -3-fluoropiperidine-1-carboxylate;
Isopropyl (3R, 4S) -4-{[6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] oxy} -3 -Fluoropiperidine-1-carboxylate 1-methylcyclopropyl (3S, 4R) -4-{[6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5 -Methylpyrimidin-4-yl] oxy} -3-fluoropiperidine-1-carboxylate;
1-methylcyclopropyl (3R, 4S) -4-{[6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidin-4-yl] Oxy} -3-fluoropiperidine-1-carboxylate; or tert-butyl 4- (6- (2,3-dihydro-1H-imidazo [1,2-b] pyrazol-1-yl) -5-methylpyrimidine -4-yloxy) piperidine-1-carboxylate or a pharmaceutically acceptable salt thereof.   8. A pharmaceutical composition comprising a compound according to any of claims 1 to 7 present in a therapeutically effective amount admixed with at least one pharmaceutically acceptable excipient.   9. The composition of claim 8, further comprising at least one additional agent selected from the group consisting of anti-obesity agents and anti-diabetic agents. The anti-obesity drug is zirlotapide, mitrapapide, imipritapide, R56918 (CAS No. 403987), CAS No. 913541-47-6, lorcaserine, cetiristat, PYY _3-36, naltrexone, oleoyl-estrone, obinetide, pramlintide, tesofensin, leptin 10. The composition of claim 9, wherein the composition is selected from the group consisting of:, liraglutide, bromocriptine, orlistat, exenatide, AOD-9604 (CAS number 221231-10-3), and sibutramine.   The anti-diabetic drug is metformin, acetohexamide, chlorpropamide, diabinase, glibenclamide, glipizide, glyburide, glimepiride, gliclazide, glipentide, glyquidone, glisolamide, tolazamide, tolbutamide, tendamistat, trestatin, acarbose, adiposine, camiglibos, Emiglitate, miglitol, voglibose, pradimicin Q, salvostatin, baraglitazone, ciglitazone, darglitazone, englitazone, isaglitazone, pioglitazone, rosiglitazone, troglitazone, exendin 3, exendin 4, trodaschemin, reserbatol, hiruthiosarp extract , Vildagliptin, alogliptin, and saxagliptin It is selected from the composition of claim 9.   A method of treating diabetes comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any of claims 1-7.   A method of treating a metabolic or metabolic related disease, condition or disorder comprising administering to a patient a therapeutically effective amount of a compound according to any one of claims 1-7.   Hyperlipidemia, type I diabetes, type II diabetes, idiopathic type I diabetes (type Ib), adult latent autoimmune diabetes (LADA), early-onset type 2 diabetes (EOD), juvenile atypical diabetes ( YOAD), juvenile-onset diabetes mellitus (MODY), malnutrition-related diabetes, gestational diabetes, coronary heart disease, ischemic stroke, restenosis after angioplasty, peripheral vascular disease, intermittent claudication, myocardial infarction (eg, necrosis) And apoptosis), dyslipidemia, postprandial lipemia, impaired glucose tolerance (IGT) state, fasting plasma glucose abnormal state, metabolic acidosis, ketosis, arthritis, obesity, osteoporosis, hypertension, congestive heart failure, left ventricular hypertrophy, Peripheral arterial disease, diabetic retinopathy, macular degeneration, cataract, diabetic nephropathy, glomerulosclerosis, chronic renal failure, diabetic neuropathy, metabolic syndrome, syndrome X, Pre-syndrome, coronary heart disease, angina pectoris, thrombosis, atherosclerosis, myocardial infarction, transient ischemic attack, stroke, vascular restenosis, hyperglycemia, hyperinsulinemia, hyperlipidemia Hypertriglyceridemia, insulin resistance, impaired glucose metabolism, impaired glucose tolerance, fasting plasma glucose abnormalities, obesity, erectile dysfunction, skin and connective tissue disorders, foot ulceration and ulcerative colitis, endothelium Selected from the group consisting of disorders and vascular disability disorders, hyperapo B lipoproteinemia, Alzheimer's disease, schizophrenia, cognitive impairment, inflammatory bowel disease, ulcerative colitis, Crohn's disease, and irritable bowel syndrome A method for the treatment of a disease, condition or disorder comprising the administration of a therapeutically effective amount of a compound according to any of claims 1-7. A method of treating a metabolic or metabolic related disease, condition, or disorder, in a patient in need of such treatment,
(I) a first composition according to claim 8, and (ii) at least one additional agent selected from the group consisting of anti-obesity agents and anti-diabetic agents and at least one pharmaceutically acceptable agent. Administering two separate pharmaceutical compositions comprising a second composition comprising an excipient.   16. The method of claim 15, wherein the first composition and the second composition are administered simultaneously.   16. The method of claim 15, wherein the first composition and the second composition are administered sequentially in any order.   Use of a compound according to claims 1 to 7 in the manufacture of a medicament for treating a disease, condition or disorder that modulates the activity of the G protein coupled receptor GPR119.   Use of a compound according to any of claims 1 to 7 in the preparation of a medicament for the treatment of diabetes or pathological conditions associated with said diabetes.

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